[26]

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

591

being implemented. Nevertheless, a great deal of effort is being given to designing the computer program, CLOGP, to assure sufficient flexibility to take care of future needs, while still maintaining a user friendliness which quantum chemical methods will find difficult to match. As supportive as we are to the idea of calculating log eoct/water from structure, we are totally opposed to using these values in reports for technical journals unless the calculation methodology is documented. All too frequently a table of values is referenced merely, "Calculated by Rekker's (or Leo's) methods," but even someone very familiar with those methods may find it impossible to duplicate the published results. Details of each calculation might be too lengthy for most publications, but the most significant one could be chosen as an example. In this respect, calculation by computer has many distinct advantages. The format of the output is standardized, and will become familiar to those following this field. If the version used is stated, one can become aware of any recent improvements and can more easily reconcile any apparent discrepancies. Also there is a trap in manual calculations: If a calculated value fits into the pattern one expected to see, there is a tendency not to check it quite as carefully for errors or omissions. A computer program may have flaws, but it delivers the same result each time, and can save one from falling into the trap.

[26] E n z y m a t i c C a t a l y s i s in O r g a n i c S y n t h e s i s

By C.-H. WONG, G.-J. SHEN, R. L. PEDERSON, Y.-F. WANG, and W. J. HENNEN Introduction Of the more than 2000 enzymes isolated, approximately 50 have been utilized for organic synthesis. The perception that enzymes are too expensive and too specific to be of general use in organic synthesis is no longer valid. Many enzymes have been shown to catalyze transformation of unnatural substrates to products of synthetic value.l-3 Enzyme-catalyzed organic reactions have been extended from the synthesis of chiral synthons and low molecular weight substances such as sugars and peptides to more I C.-H. Wong, Science 244, 1145 (1989). 2 j. B. Jones, Tetrahedron 42, 3351 (1986). 3 E. J. Toone, E. S. Simon, M. D. Bednarski, and G. M. Whitesides, Tetrahedron 45, 5365 (1989).

METHODS IN ENZYMOLOGY, VOL. 202

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

592

ENZYMES

[26]

complex molecules such as oligosaccharides, polypeptides, nucleotides, and their conjugates. All recombinant DNA work today requires several key enzymatic reactions to construct the gene for expression of a desired protein. The recombinant DNA technology, however, has made possible the low-cost production of enzymes and the rational alteration of enzymatic properties. The area of enzymatic catalysis is further stimulated by the exciting new discovery of catalytically active antibodies. 4-8 With the increasing environmental concerns and regulatory constraints faced in the chemical and pharmaceutical industries, enzyme-based organic synthesis becomes an attractive alternative which may offer clean and mild catalytic processes for the synthesis of single stereoisomers. This chapter describes the fundamental concepts and the practical aspects regarding the design and development of enzymatic catalysts for synthetic organic transformations. Rate Acceleration and Specificity of Enzymatic Reactions The rate acceleration and specificity of enzymatic reactions which operate under mild conditions (e.g., at room temperature and neutral pH) are the major advantages of enzymes used in organic synthesis. According to transition state theory9 and the thermodynamic cycle, one can conclude that in a given enzyme-catalyzed reaction the catalyst binds to the reaction transition state more strongly than to the ground state substrate by a factor approximately equal to the rate acceleration. 10-13 All types of catalysis in enzymatic reactions, such as acid-base catalysis, nucleophilic-electrophilic catalysis, and catalysis by approximation, strain, and distortion are just the contributing factors that lead to reducing the transition state energy. 14 Figure 1 illustrates the difference between enzyme-catalyzed and uncatalyzed reactions. This concept of transition state stabilization in enzymecatalyzed reactions has been experimentally confirmed and applied to the design of new catalysts and new enzyme inhibitors. Many potent enzyme inhibitors are transition state analogs which bind to enzymes much more 4R. A. Lerner, Proc. Solvay Conf. Chem. 18th, p. 42 (1983). 5A. Tramontano, K. D. Janda, and R. A. Lemer, Science 2;34, 1566(1986). 6 S. J. Pollack, J. W. Jacobs, and P. G. Schultz, Science 234, 1570(1986). 7D. Hilvert and K. D. Nared, J. Am. Chem. Soc. U0, 5593 (1988). s R. A. Lerner and S. J. Benkovic, Chemtracts: Org. Chem. 3, 1 (1990). 9 H. Eyring,J. Chem. Phys. 3, 107 (1935). 10L. Pauling,Am. Sci. 36, 51 (1948). 11j. L. Kurz, J. Am. Chem. Soc. 85, 987 (1963). 12R. Wolfenden,Nature (London) 223, 704 (1969). 13G. E. Lienhard,Annu. Rep. Med. Chem. 7, 249 (1972). 14j. Kraut, Science 242, 533 (1988).

[26]

ENZYMATIC

CATALYSIS

IN ORGANIC

•

-"

~I > . .®-

,

u~

o.

4-

-l-

U.l

II

LU

%

+

~

tJ'}

ILl

LI.I w

0 0

m 0

I"

0

G)

0

E

s~

m ¢.0

'I~

=

tJ) 4-

~ ¢J)

tJ'}

ILl

e=

~)

I--

I.-

u6 •

@

594

ENZYMES

[26]

strongly than do substrates, products, intermediates, or their analogs.12-17 The enzymatic functional groups interacting with a transition state analog as indicated in the X-ray crystal structure ~7'~8of the complex are considered to be those involved in transition state stabilization. With regard to specificity, enzymes accept certain type of substrates. E n z y m e s are selective not only in the type of reaction they catalyze, but also in their position of attack on a molecule. 1-3 Chemoselective reaction of one of different functional groups in a molecule, regioselective reaction of one of the same or similar groups in a molecule, enantioselective reaction of one enantiomer o f a racemic pair, and enantiotopic or diastereotopic group or face attack of a chiral or prochiral molecule are all well documented. All such stereoselectivities originate from the energy difference in the e n z y m e - t r a n s i t i o n state complex. Figure 2 illustrates the use of steady-state kinetics for the analysis of an enantioselective transformation where A and B compete for the active site of enzyme. The enantioselectivity of the reaction is determined by the ratio of the specificity constraints, k c a t / K m, of the two competing reactions and is related to the free energy term hAG*, the difference in energy of diastereomeric transition state complexes. ~9 In an enzyme-catalyzed enantioselective transformation of a racemic mixture, the enantioselectivity can be related to the extent of conversion (C) and the enantiomeric excess (ee) of the product or unreacted substrate. The enantioselectivity value E ' , once determined, can be used to predict the ee value of the unreacted substrate or the r e c o v e r e d product at a certain degree of conversion. 2°

Engineering E n z y m e Specificity As indicated in Table I, the energy difference required to obtain a 99.9% ee product from an enzymatic reaction is relatively small, approximately 4.5 kcal/mol. A simple way to improve the enantioselectivity o f an e n z y m e reaction is to change the reaction conditions (such as lowering or raising the temperature, or adding organic solvent or other substances) so that the energies of diastereomeric e n z y m e - t r a n s i t i o n state complexes are altered favorably.~ Another way to increase the magnitude of AAG* is 15W. P. Jencks, "Catalysis in Chemistry and Enzymology." McGraw-Hill,New York, 1969. 16p. A. Bartlett and C. K. Marlowe, Biochemistry 22, 4618 (1983). 17T. L. Blundell, J. Cooper, S. I. Foundling, D. M. Jonex, B. Atrash, and M. Szelke, in "Perspective in Biochemistry" (H. Neurath, ed.), Vol. 1, p. 84. American Chemical Society, Washington, D.C., 1989 18D. E. Tronrud, N. M. Holden, and B. W. Matthews, Science 235, 571 (1987). t9 A. R. Fersht, "Enzyme Structure and Mechanism." Freeman, New York, 1985. 20C.-S. Chen, Y. Fujimoto, G. Girdaukas, and C. J. Sih, J. Am. Chem. Soc. 104, 7294(1982).

[26]

595

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

EA

(k©,,t)A

(kcat)e

E + A

( kcat/Km)A ~ q

~,,

E+

,.

EA ~

P

E+Q

(kcat/Km)B

E + B

EB ~

AG A~

•

A G a ;~

EB ~

AG

E+Q

VA --= VB

(kcat/Km) A [A]

E+P

(kcat/Km)A

,

(kcat/Km)B [B]

In [(1 - c)(1 - e e e ) ] In [(1 - c)(1 + e e e ) ]

- e x p ( - A A G * / R T ) = E'

(kcat/Km) B

In [1 - c(1 + eep)] =

In [1 - c(1 - eep)]

= E'

FIG. 2. Enzyme-catalyzed enantioselective reactions with a racemic mixture (A + B). E, Enzyme; P, product from A; Q, product from B; E ', enantioselectivity value; c, percent conversion; eep, product ee; ee B, ee of recovered B.

to change the substrate structure by introducing a removable protecting group.' For enzymes with known X-ray structure, the techniques of sitedirected mutagenesis and computer-assisted molecular modeling can be used to rationally alter or improve enzyme specificity and catalysis. For example, aspartate aminotransferase was converted to lysine-arginine aminotransferase by replacing the active site Arg with Asp. 21 L-Lactate 21 C. N. Cronin, B. A. Malcolm, and J. F. Kirsch, J. Am. Chem. Soc. 109, 2222 (1987).

596

ENZYMES

[26]

TABLE I ENERGY REQUIRED FOR CALCULATED ENANTIOMERIC EXCESS IN GIVEN ENZYMATIC REACTION Enantiomeric excessa (% ee)

(kcat/Km)A/(kcat/Km) B

A A G *(kcal/mol)

10 50 90 95 99 99.9

1.22 3 19 39 199 1999

0.118 0.651 1.74 2.17 3.14 4.50

a % ee = [(kcat/Km) A -- (kcat/Krn)B]/[(kcat/Km) A + (kcat/Km)B].

dehydrogenase was converted to L-malate dehydrogenase. 22 The serine protease subtilisin BPN' has been engineered to alter the catalysis and specificity. 23 The technique of site-directed mutagenesis, however, is still quite limited. So far, no drastic change of specificity (e.g., from D to L substrates) has been successful. For this reason, traditional screening is still a very economical and efficient way of finding enzymes for desirable reactions. A bottleneck in screening, however, is the time-consuming process involved in the selection and assay for enzyme activities. New strategies (e.g., positive or negative selection with designed substrates, or selection with substrates containing chromogenic or pH-sensitive leaving groups) must be employed to facilitate the process. Once an enzyme is identified, the gene can be cloned and overexpressed. One problem in enzymatic reactions is the difficulty of predicting the stereochemistry of a reaction. Although the stereoselectivity of an enzyme is generally the same and is controlled by the unique tertiary structure of the catalyst, a changeover in stereoselectivity from one substrate to another is not uncommon. This changeover represents a significant problem for many structurally unknown enzymes. The only solution at this stage is to develop a reliable active site model based on computer graphic analysis of the substrate-specificity results. 24 A useful model must be simple and reliable, and it can be used in the prediction of new reactions and in the rationalization of published results. 22 H. M. Wiks, K. W. Hart, R. Feeney, C. R. Dunn, H. Muirhead, W. N. Chia, D. A. Barstow, T. Atkinson, A. R. Clarke, and J. J. Holbrook, Science 242, 1541 (1988). 23 j. A. Wells and D. A. Estell, Trends Biochem. Sci. (Pets. Ed.) 291 (1988). 24 I. J. Jakovac, H. B. Goodbrand, K. P. Lok, and J. B. Jones, J. A m . C h e m . Soc. 104, 4659 0982).

[26]

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

597

Issues of Enzyme Stability Enzymes are intrinsically unstable in solution and can be inactivated by several processes including denaturation (often caused by a change of temperature, pH, or dielectric environment), cofactor dissociation, proteolysis, and oxidation. The three-dimensional structure of an enzyme in solution is determined by its primary sequence and is the one with the l o w e s t A G . 25 Enzyme inactivation often occurs through reversible partial denaturation followed by irreversible changes. Thermophilic enzymes are usually more stable than mesophilic enzymes. The former usually retain their native conformation between 55 ° and 70 °, and the latter at temperatures below 40 °. Thermophilic enzymes, however, differ from their mesophilic counterparts only by small changes in primary structure. 26 The three-dimensional structures are essentially the same. This temperature difference corresponds to 5-7 kcal/mol as determined by the rate of inactivation as a function of temperature. Free energy changes of this order can be derived from a few additional salt bridges, hydrogen bonds, or aromatic-aromatic interactions. Unstable enzymes therefore could be made more stable by introducing additional binding forces by site-directed mutagenesis or chemical modifications, provided such modifications do not perturb the active conformation of enzyme. A subtilisin mutant, for example, prepared recently via multiple site-specific mutations is several hundred times more stable than is the wild type in aqueous solution and 50 times more stable in dimethylformamide. 27 A further improvement of this variant resulted in a 1000-fold increase in stability in dimethylformamide. Enzyme immobilization via a multiple point attachment to a solid support is presently the most commonly used technique for enzyme stabilization. 2s The procedures generally involve covalent or noncovalent attachment of enzymes to a support. Cross-linking and entrapment or encapsulation are also often used. Glass beads treated with 3-aminopropyltriethoxysilane, a cross-linked copolymer of acrylamide and acryloxysuccinimide, or epoxide-containing acrylamide beads (Eupergit C, New York) are often used as solid support for covalent immobilization. 25 C. B. Anfinsen, Science 181, 223 (1973). 26 V. V. Mozhaev and K. Martinek, Enzyme Microb. Technol. 6, 50 (1984). 27 c . - n . Wong, S. T. Chen, W. J. Hennen, J. A. Bibbs, Y.-F. Wang, J. L.-C. Liu, M. W. Pantoliano, M. Whitlow, and P. N. Bryan, J. Am. Chem. Soc. 112, 945 (1990); Z. Zhong, J. L. C. Liu, L. M. Dintermann, M. A. J. Finkelman, W. T. Mueller, M. L. Rollence, M. Whitlow, and C.-H. Wong, J. Am. Chem. Soc. 113, 2259 (1991). 28 K. Mosbach (ed.), this series, Vols. 135-137.

598

ENZYMES

[26]

Ion-exchange resins, glass beads, and XAD-8 are often used for adsorption of enzymes to be used in organic solvent or biphasic systems.

Cofactor Regeneration Many synthetically useful enzymatic reactions require cofactors such as adenosyl triphosphate (ATP), nicotinamide adenine dinucleotidc (NAD), and its 3'-phosphate (NADP), acetyl coenzyme A (AcCoA), Sadenosylmethionine (SAM), and 3'-phosphoadenosine- 5'-phosphosulfate (PAPS). These cofactors are too expensive to be used as stoichiometric reagents. Regeneration of the cofactors from their reaction by-products is required to make the process economical. 29'3° Cofactor regeneration can also (1) influence the position of equilibrium, that is, a thermodynamically unfavorable reaction can be driven by coupling with a favorable cofactor regeneration reaction, and (2) prevent the accumulation of cofactor byproduct which may inhibit the forward reaction and complicate the workup procedure. The cofactor ATP and other nucleoside triphosphates have been used concurrently with regeneration of the cofactors for selective enzymatic phosphorylations. 1,3,3° It is now possible to synthesize sugar phosphates, nucleotides, oligosaccharides, and related substances in large quantities with cofactor regeneration. Glycosyltransferases coupled with enzymatic cofactor regeneration, for example, have been used for the synthesis of glycopeptides and oligosaccharides associated with tumor development and various biochemical recognitions.l Stoichiometric glycosidation based on glycosyltransferases is also a valuable approach to glycoprotein remodeling. Recombinant glycoproteins with heterogeneous carbohydrate structures may be modified to homogeneous species via glycosidase and glycosyltransferase reactions (Fig. 3). For nicotinamide cofactor-dependent reductions, formate-formate dehydrogenase31 and glucose-glucose dehydrogenase32 are the most useful regeneration systems for NADH, and glucose-glucose dehydrogenase32 and 2 - p r o p a n o l - T h e r m o a n a e r o b i u m brockii are suitable for NADPH. 33 For oxidation reactions, ketoglutarate-glutamate dehydrogenase for re29H. K. Chenaultand G. M. Whitesides,Appl. Biochem. Biotechnol. 14, 147 (1987). 3oH. K. Chenault,E. S. Simon,and G. M. Whitesides,Biotechnol. Genet. Eng. Rev. 6, 221 (1988). 31R. Wichmann,C. Wandrey,A. F. Buckmann,and M. R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 32C.-H. Wong, D. G. Drueckhammer, and H. M. Sweers, J. Am. Chem. Soc. 107, 4028 (1985). 3t R. J. Lamedand J. G. Zeikus,Biochem. J. 195, 183 (1981).

[26]

ENZYMATIC CATALYSISIN ORGANICSYNTHESIS

SialCMrPanNsleU~c 1t, ~

GI[NAc (Ser) I GIcNAc CO2"

599

GI[NAc (Ser) GllcNAc N ~ o - N - a eet YIgly eOsaminidase H

[ (Ser) GIcNAc~I UDP-GlcNAc [ GDP-Man [ Glycosyl transferase~

I

(Ser)

Man!31,4GIcNAcl31,4GIcNAcl31 Man~'% FIG. 3. Glycoprotein remodeling involving removal of undesired sugars with glycosidases and addition of desired sugars with glycosyltransferases.

generation of NAD or NADP and pyruvate-lactate dehydrogenase for NAD are considered the most u s e f u l. 29 The system based on flavin mononucleotide (FMN) and FMN reductase 34is potentially useful. A new NADdependent alcohol dehydrogenase recently isolated from Pseudomonas species is useful for the synthesis of chiral secondary alcohols and for the regeneration of N A D H using 2-propanol as hydride donor. 35 This enzyme possesses a different stereospecificity compared to the other known alcohol dehydrogenases (Fig. 4). When a second enzyme is used for cofactor s4 D. G. Drueckhammer and C.-H. Wong, J. Org. Chem. 50, 5387 (1985). 35G.-J. Shen, Y.-F. Wang, C. Bradshaw, and C.-H. Wong, J. Chem. Soc., Chem. Commun. 677 (1990).

600

ENZYMES

[26]

E2

ADPR

ADPR

FIG. 4. Stereospecificity of a new alcohol dehydrogenase isolated from Pseudomonas. S and L indicate small and large substituents, respectively. Enzyme sources: El, Pseudomonas; E2, Mucorjaoanicus; E 3, yeast, horse liver, and Thermoanaerobium.

regeneration, the regeneration system, in some instances, can be synthetically useful if the overall reaction is favorable and the products are easily separated. For example, a one-pot biphasic synthesis of a lactone and an amino acid was carried out in which NAD-dependent oxidation of a m e s o diol was coupled with NADH-dependent reductive amination.l,36 The biphasic system has another advantage, namely, that product inhibition is minimized, thus the overall yield is increased. In addition to the nicotinamide cofactor-dependent oxidoreductions with direct hydride transfer between the substrates, there are many oxidoreductases such as oxygenases which use N A D H or NADPH as indirect reducing agents.~ For synthetic transformations, whole cells instead of isolated oxygenases are used because of the instability of the enzymes. Cofactor SAM is generally involved in selective methylation, and PAPS is involved in selective sulfation. Currently there is no regeneration system available for enzymatic reactions requiring these two cofactors. Effect of Organic Solvent Organic solvents are often added to aqueous solutions of enzymatic reactions to increase the substrate solubility or to change the equilibrium. Homogeneous, biphasic, and reverse-micelle cosolvent systems have been used. Some enzymes are active and stable in monophasic organic solvent systems which contain a minimum amount of water to cover the enzyme surface. 37'38 In some instances, higher thermostability and different substrate specificities of enzymes have been observed in transitions from water to organic solvents. Enzyme-catalyzed dehydrations, transesterifi36 j. R. Matos and C.-H. Wong, J. Org. Chem. $1, 2388 (1986). 37 A. M. Klibanov, Trends Bioehem. Sei. (Pers. Ed.) 14, 141 (1989). 38 C. S. Chen and C. J. Sih, Angew. Chem., Int. Ed. Engl. 28, 695 (1989).

[26]

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

601

cations, aminolyses, and oxidoreductions in monophasic organic solvents are common. Novel enzymatic reactions in gases and supercritical fluids have also been reported. Product or substrate inhibition can be lessened if the products or substrates are retained mostly in the organic phase. The reaction is usually done with shaking. After the reaction is complete, the enzyme can be recovered through filtration or centrifugation and used repeatedly. The enzyme can also be immobilized to improve the stability and to facilitate the recovery. Organic solvents, however, are not generally useful media for enzymatic reactions, because (1) they are not applicable to compounds insoluble in organic solvents, (2) adjusting the pH of the reaction is difficult, (3) severe substrate or product inhibition may occur for enzymatic reactions with hydrophilic substrates such as sugars, (4) many organic solvents are not environmentally acceptable, and (5) loss of stereoselectivity may occur owing to the effect of organic solvent or the reverse nature of the reaction. Taking the example of esterase-catalyzed transesterification for the kinetic resolution of a racemic alcohol, if the o-isomer is a better substrate than the L-isomer, accumulation of the D-ester and the unreacted L-alcohol will occur in the forward reaction. In the reverse reaction, however, the o-ester is a better substrate and is converted to the D-alcohol according to the principle of microscopic reversibility. The enantiomeric excesses of both the o-ester and the L-alcohol therefore decrease progressively as the extent of the reverse reaction increases. Product inhibition caused by the released alcohol is another concern. These problems can be overcome by the use of enol esters as transesterification reagents. The leaving groups of enol esters tautomerize to ketones or aldehydes that are volatile and do not undergo the reverse reaction. 39'4° Furthermore, the kinetics for irreversible reactions are easier to handle than the reversible cases. The combination of transesterification and hydrolysis thus provides a simple way for the preparation of both enantiomers. For example, both (R)- and (S)-2-O-benzylglycerol monoester can be prepared from 2-O-benzylglycerol and the diacetate via lipase-catalyzed transesterification and hydrolysis, respectively 39 (Fig. 5). The enzyme possesses the same enantiotopic group selectivity in each case. Extension of this concept to the synthesis of nucleosides using 7-methylguanosine as an irreversible transribosylation reagent has been demonstrated. 41 Since many enzymatic transformations are carded out in unnatural 39 Y.-F. Wang and C.-H. Wong, J. Org. Chem. 53, 3127 (1988). 4o Y.-F. Wang, J. J. Lalonde, M. Momongan, D. E. Bergbreiter, and C.-H. Wong, J. Am. Chem. Soc. 110, 7200 (1988). 41 W. J. Hennen and C.-H. Wong, J. Org. Chem. 54, 4692 (1989).

602

ENZYMES OCH2Ph HO~,~O H

O "Jl'oJl~ Lipase-

OCH2Ph "W'°""~°'W o

o

"

H20 Lipase

[26]

OCH:~h .~,,O,~OH O

0

+

,~

OCH'~h I

)

0

-

Ov,kvoy-

+

.,,x. o H

o

FIG. 5. Transesterification using an enol ester to obtain one enantiomer. Hydrolysisof the meso-diester gives the other enantiomer. environments such as organic solvents with unnatural substrates, the enzymes are not optimized with regard to catalysis and stability. The technique of site-directed mutagenesis may find use in terms of improving enzyme catalysis and stability. The development of a mutant subtilisin as described previously represents a typical example of engineering enzymes for synthetic applications. Catalytic Antibody As mentioned before, one mechanism by which enzymes act as highly specific catalysts is to provide steric and electronic complementarity to a rate-determining transition state of a given reaction.l° This concept leads to the suggestion that an antibody to a stable transition state analog of a reaction should catalyze that reaction. ~5Today, several catalytically active antibodies have been developed for stereocontrolled reactions with rate accelerations of several orders of magnitude, some even approaching that of enzyme catalysis. 5-8 With appropriate design of antigens, functional groups can be induced in the catalytic site of an antibody to perform general acid-base or nucleophilic-electrophilic catalysis in addition to overcoming the entropic barrier in catalysis. With this technique, new protein catalysts can be designed and prepared for reactions which may not exist in nature. This new development changes our perception regarding the design of catalysts for chemical reactions. A bottleneck in the rapidly evolving field of antibody catalysis, however, is the inefficiency and inaccessibility of the hybridoma technology for the production of desired monoclonal antibodies in large quantities for chemical transformations. Further, the number of antibodies induced to a synthetic antigen based on the hybridoma technology is quite limited (only

[26]

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

603

a few hundred antibodies are generated), and the methods used to detect the antigen binding are not efficient. The antibodies produced, therefore, may not represent the whole group of antibodies induced to a given antigen. The probability of finding the desired antibodies for catalysis is thus relatively low. Furthermore, if the synthetic antigen does not closely resemble the transition state of the reaction, the antibodies induced may not have appropriate functional groups to participate in catalysis. Part of the problem has been solved by the creation of a highly diverse library of heavy and light chain antigen-binding fragments (Fab's) for use in screening for the desired Fab. 42 The methodology depends on the use of the polymerase chain reaction (PCR) in the presence of designed DNA primers to amplify the mRNAs from immunized mice spleens followed by cloning into h phage and construction of a plasmid expression library in Escherichia coli. Since Fab fragments behave as whole antibodies in terms of antigen recognition, and since site-directed mutagenesis of the complementarity determining regions (CDRs) of Fab's can be easily carried out to improve the binding and catalysis, the field of catalytic antibodies based on the Fab fragments will experience a substantial development in the future. A detailed and improved procedure for the construction of Fab libraries from immunized mice is described below.

Selected Examples

Engineering Serine Proteases to Peptide Ligases

Proteases have been useful as catalysts in peptide synthesis because the reactions are catalytic, regio- and stereoselective, racemization-free, and require minimal side-chain protection under mild reaction conditions. 43-46Both thermodynamic approaches (i.e., direct reversal of hydrolysis) and kinetic approaches (i.e., aminolysis) have been used. The kinetic approach is faster, but the amidase activity of proteases tends to cleave sensitive peptide bonds in the substrates or products, which results in miscoupling (transpeptidation) and undesirable peptide hydrolysis. These undesired reactions can be eliminated or lessened when N ~ of the active42 W. D. Huse, L. Sastry, S. A. Iverson, A. S. Kang, M. Alting-Mees, D. R. Burton, S. J. Benkovic, and R. A. Lerner, Science 246, 1275 (1989). 43 j. S. Fruton, Adv. Enzymol. 53, 239 (1982). 44 W. J. Kullmann, Protein Chem. 4, l (1982). 45 K. Oyama, K. Kihara, Chem. Tech. (Leipzig) 14, 100 (1984). 46 K. Morihara, Trends Biotechnol. $, 164 (1987).

604

ENZYMES

[26]

site His is methylated 47'48or when certain amounts (50-70%, v/v) of watermiscible organic solvents (e.g., dimethylformamide, dioxane, or acetonitrile) are added to the aqueous enzyme solution. 27'49 Mechanistic investigation indicates that the methylated imidazole may undergo ring flipping in order to act as a general base for aminolysis. Analysis of the reaction kinetics reveals that the transition state energy required for ester hydrolysis increases more than that for aminolysis. Therefore, the energy barrier for the formation of acyl intermediate-nucleophile complex for aminolysis is lower. In addition, the affinity of the peptide product for the methylated enzyme increases for charged or hydrophilic products and decreases for hydrophobic products. Energy diagrams for reactions catalyzed by both native and methylated chymotrypsin are shown in Fig. 6. Similar situations are observed in the case of organic cosolvent-mediated catalysis. When native serine proteases are placed in pure water-miscible organic solvents, either the enzymes are inactivated or the reactions are too slow to be useful for synthesis.

Enzymatic Aldol Condensations Aldolases are a group of enzymes proven to be useful for the synthesis of sugars and related substances. 3'5° Of more than 20 aldolases reported, 8 have been used for organic synthesis. Two common features are found in aldolase-catalyzed reactions: the enzymes are specific for the donor substrate but flexible for the acceptor substrate, and the stereochemistries of the aldol addition reactions are controlled by the enzymes, not by the substrates. This broad acceptor specificity permits the use of this class of enzymes for the synthesis of novel sugars. Several microbial aldolases have recently been cloned, overexpressed, and applied to synthesis) 1'52 For example, we have developed an effective strategy for the synthesis of piperidines structurally related to monosaccharides. Deoxynojirimycin

47 j. B. West, J. Scholten, W. J. Stolowich, J. L. Hog,g, A. I. Scott, and C.-H. Wong, J. Am. Chem. Soc. 110, 3709 (1988). 48 j. B. West, W. J. Hennen, J. A. Bibbs, J. J. Lalonde, Z. Zhong, E. F. Meyer, and C.-H. Wong, J. Am. Chem. Soc. 112, 5313 (1990). 49 C. F. Barbas III, J. R. Matos, J. B. West, and C.-H. Wong, J. Am. Chem. Soc. 110, 5162 (1988). 5o G. M. Whitesides and C.-H. Wong, Angew. Chem., Int. Ed. Engl. 24, 617 (1988). 51 C. H. v o n d e r Osten, A. J. Sinskey, C. F. Barbas III, R. L. Pederson, Y.-F. Wang, and C.-H. Wong, J. Am. Chem. Soc. 111, 3924 (1989). 52 C. F. Barbas III, Y.-F. Wang, and C.-H. Wong, J. Am. Chem. Soc. 112, 2013 (1990); A. OzaRi, E. J. Toone, C. H. Von der Osten, A. J. Sinskey, and G. M. Whitesides, J. Am. Chem. Soc. 112, 4970 (1990).

[26]

605

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

0

II B o c N H ~ C - O H

O II BocNH-~mmv--C-OR'

O II

E ~

(P0

R--C--OE

(ES')

/'/2~'~.l,s,:~.v~

O II

s e r i n e protP~lses

Native

......

~]l/d~ "'ll"lll~*~'*~ S l { ~ N I I ~

Serine proteases methylated at N E of the active-site His

~NH'I

~- R "

(P2)

AAG = 5.2 kcal/mol•

'~

I Ii

II l|

i i i i

.

"6

i i I

~

I I

:~ ' ~ AAG = 6.lkeal/mol •

!

it

q~

"=

E:P2 E + P2

(Peptide)

AAG -0.6 k ~ / m o l

ES' AAG = 2 keal/mol

E + P1 (acid)

Reaction progress FIG, 6. Active-site directed modification of serine proteases to peptide ligases.

and deoxymannojirimycin, for example, have been prepared based on a combined aldol condensation and reductive amination 51 as shown in Fig. 7. The aldehyde substrates were prepared via a lipase-catalyzed resolution of 2-acetoxy-3-azidopropanal diethyl acetal. The donor substrate dihydroxyacetone phosphate (DHAP) can be replaced with a mixture of dihydroxyacetone and a small amount of arsenate. This strategy perhaps can be extended to the synthesis of piperidines structurally related to other sugars such as sialic acid, fucose, rhamnose, and galactose with the use of other aldolases. Synthesis of Dihydroxyacetone Phosphate. The synthetic value of DHAP-dependent aldolases depends on the availability of DHAP. Several methods for the synthesis of this compound are available. 3 The following

606

ENZYMES I

oI

H~ /FDP

[26]

N3 OH ~. Aldolase

0

OH "

2"O3PO

OH

O

. 1) PhosphataDse N3 2) H2/ Pd HO

H I

OH

OH Deoxynojirimycin

2O3PO~,,~fOH FDP Aldolase

o

OH

~ 20aPO

O H

N3

OH

N3

OH

l) Phosphat~e 2) H2/ Pd

OH

H

NH H

HO Deoxymannojirimyci

OCOCH3 N3

OC2H$ OC2Hs

LP.80-Lipase

N3

OCOCH3 .-• OC2H$ OC2Hs • 98% ee

OH +

N3

OC2H$ OC2Hs • 97% ee

FIG. 7. Combined FDP (fructose-1,6-diphosphate) aldolase reaction and reductive amination for the synthesis of aza sugars.

describes an improved procedure based on a trivalent phosphorylating reagent we have recently developed (Fig. 8). 2,5-Diethoxy-p-dioxane-2,5-dimethanol. A modified procedure of Fischer and Mildbrand 53 is used for the synthesis of 2,5-diethoxy-p-dioxane2,5-dimethanol. To an oven-dried l-liter round-bottomed flask is added 400 ml of anhydrous ethanol, 1.84 g concentrated H E S O 4 ( 1 8 . 8 mmol), and 31.12 g (210 mmol) of triethyl orthoformate; this solution is refluxed for 30 min under N 2. The solution is cooled at 4°, and 1.41 g (7.8 mmol) of dry dihydroxyacetone dimer (DHA) is added every 12 hr for 72 hr. After the last addition, the solution is stirred for an additional 24 hr at 4 °. Water (4 ml) is added, and the mixture is stirred for 30 min. The pH of the solution is adjusted to 8.0 with 2 N NaOH, and the excess ethanol is removed under reduced pressure. The white precipitate is triturated with 2 x 100 ml ether; the ether fractions are combined and extracted with 20 ml of saturated NaC1. The aqueous fraction is extracted with ether (3 x 40 ml). The combined ether fractions are dried over anhydrous Na2SO 4 and evaporated under reduced pressure. The product is recrystallized from ethyl acetate/ heptane to produce 2.6 g of the title compound. The white precipitate from the ether triturations is again triturated with 100 ml of ether until product cannot be detected in the extract by thin53 H. O. L. Fischer and H. Mildbrandt, Ber. Dtsch. Chem. Ges. 57, 710 (1924).

[26]

607

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

O-/~OH

O"~

OH

b

O'~

OR C

H0.__~, 0

HO-.--•

0

RO.---.• R =

0

P(O)(OCH2Ph)2

o.~OPO3H " O EtO

2-O3PO----/

OEt -O,

3Na+

d

,,,

2

HO~OPO3

2

DHAP

FIG. 8. Synthesis of dihydroxyacetone phosphate. (a) Ethanol, H2SO4 cat, HC(OEt)3 ; (b) (PhCH20)2PNEt 2, triazole (4 equivalents), then 30% H202; (c) Pd/C, H2; (d) H +, 65°, 4 hr.

layer chromatography (TLC) (500 ml). The ether fractions are combined, then concentrated under reduced pressure, and the product is recrystallized from ethyl acetate/heptane to yield 7.1 g. Combining the two recrystallized precipitates yields 9.7 g (41.4 mmol, 87%) of the title compound. Dibenzyl-N,N-diethylphosphoramidite (DDP). To a dry 3-necked 5liter round-bottomed flask equipped with an overhead stirrer are added 4.2 liters of anhydrous ether and 137.33 g (1.0 mol) of phosphorus trichloride. The solution is cooled to 0°, under dry N2; 73.14 g (1.0 mol) diethylamine and 101.19 g (1.0 mol) triethylamine are added dropwise. The mixture is stirred at room temperature for 24 hr (efficient stirring is required for good yields). The solution is again cooled at 0°, and 216.28 g (2.0 mol) of benzyl alcohol and 220.0 g (2.17 mol) of triethylamine are added dropwise. The solution is stirred at room temperature for 36 hr. The triethylamine hydrochloride salt is removed by filtration, and the ether is removed under reduced pressure. The remaining residue is vacuum distilled (bp0.3 154°-156 °) to yield 196.5 g (0.619 mol, 62%) DDP. This compound is stable and can be kept for several months without any decomposition or disproportionation. 1H NMR (200 MHz, CDCI3) ~ 1.1 (2t, 6H, 2 CH 3, J = 7.1 Hz), 3.1 (2q, 4H, 2 NCH 2, J = 7.1 Hz), 4.75, 4.8 (2d, 4H, 2 CH_H_2Ph),7.3 (m, 10H, 2 Ph). 13C NMR (50 MHz, CDCI3)8 17.5 (CH3), 38.7, 39.1 (NCH2), 62.5, 62.9 (_CH2Ph), 123.0, 123.2, 124.1, 138.8 (aromatic).

2,5-Diethoxy-p-dioxane-2,5-dirnethanol-O-2~,O-51-Bis (phosphate) Tetrabenzyl Ester. To a 250-ml round-bottomed flask containing 100 ml of dry tetrahydrofuran (THr0, 5.9 g (25.0 mmol) 2,5-diethoxy-p-dioxane-2, 5-dimethano! and 6c~ g (100 mmol) 1,2,4-triazole are stirred under N 2. To

608

ENZYMES

[26]

the solution is added 17.43 g (55 mmol) DDP, and the solution is stirred at room temperature for 24 hr. The solution is cooled at - 7 8 ° in a dry ice/ acetone bath, and 17 ml of 30% (v/v) hydrogen peroxide is added in a single portion. The solution is allowed to warm to room temperature and is stirred for 90 min. The THF is removed under reduced pressure, 300 ml of ether is added, and the solution is extracted with 20 ml of 1 N Na2SO3, 2 x 100 ml of 1 N HCI, 100 ml saturated NaHCO3, and 100 ml water. The organic layer is dried over anhydrous N a 2 S O 4. The solvent is removed under reduced pressure to yield 17.0 g (22.5 mmol, 90%) of the title compound as a syrup. This compound is recrystallized from ether and hexane. lH NMR (200 MHz, CDC13) 8 1.15, 1.20 (2t, 6H, J = 7.1 Hz, eq/az), 3.31-4.12 (m, 12H, 2 CH3CH2, 4 CH20), 4.95, 5.05 (2s, 8H, 4 PhCH2), 7.31 (s, 20H, 4 Ph). 13C NMR (50 MHz, CDCI3) 8 15.32, 15.33, 56.57, 56.58, 63.46, 63.47, 65.46, 65.47, 65.57, 65.59, 69.42, 69.43, 69.49, 69.53, 69.54, 69.60, 93.65, 93.69, 127.97, 128.61,135.71. High-resolution mass spectroscopy (HRMS) for C38Ha6OI2P2: calculated 756.2465; observed 756.2486.

2,5-Diethoxy-p-dioxane-2 ,5-dimethanol O-21,0-51-Bis(phosphate ) Trisodium Salt. The compound prepared above is dissolved in 200 ml ethanol, then 5.0 g 10% Pd/C is added and the solution hydrogenated under 50 psi of H2 for 2 days. TLC (ether, Rf 0.4 and 0.5) indicates the absence of starting material. The mixture is filtered, and the catalyst is rinsed with 200 ml ethanol. The ethanol solution is concentrated under reduced pressure to yield a syrup. Water (300 ml) is added, the pH is adjusted to 7.2 with 5 N NaOH, and the solution is lyophilized to yield 10.98 g (20.9 mmol, 84%) of the title compound as a white powder. 1H NMR (200 MHz, D20) 8 1.2, 1.25 (2t, 6H), 3.5-3.95 (m, 12H), 4.65 (s, 6H, 3 H20). Dihydroxyacetone Phosphate. To a 100-ml round-bottomed flask containing 1.0 g (1.9 mmol) 2,5-diethoxy-p-dioxane-2,5-dimethanol 0-21,O-5 Ibis(phosphate) trisodium salt and 50 ml of water is added Dowex 50 (H +) resin ( - 2 0 ml) until the pH is 1.5. The mixture is heated at 65° for 4 hr. The resin is filtered and washed with water (2 x 20 ml). The aqueous fractions are combined, and the pH is adjusted to 3.0 with 1 N NaOH. Enzymatic assay 54 for DHAP yields 2.8 mmol (74%). Synthesis of Sugars Labeled with 13C at Adjacent Carbons. Monosaccharides labeled with 13C at adjacent carbons are useful for the study of carbohydrate metabolism. The following procedure describes the synthesis of DHAP ~3C-labeled at the C-2 and C-3 positions. This compound should be useful for the synthesis of 13C-labeled monosaccharides based on aldolase reactions. 54 H. U. Bergmeyer, "Methods of Enzymatic Analysis," 3rd Ed., Vol. 2, p. 146. Verlag Chemie, Deerfield, Florida, 1984.

[26]

609

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS 0

Br

OH

PhCH20 H ~

a

0 PhCH20.,~O

•

PhCH20

S S

/lt

MeO OMe PhCH20~,,,~OH

c

0 H

d

MeO OMe ~l --- P h C H 2 0 ~ , , , . ~ O p _ OPh

e

OPh 2

3 /

_O. HOw.

v OPO32"

4

r---OH

O

~

OH

t~==O OH

OH

OH

5a

5b

CH2OH

+

$

OH

CH2OH 5c

FIG. 9. Synthesis of [2,3-13C]dihydroxyacetone phosphate and o-[2,3-13C]xylulose. (a) NaH/Bu4NI; (b) CH2N2, then lithium dithiane; (c) 1, trimethyl orthoformate/H+; 2, CH3I/ CaCO3, then NaBH4; (d) (PhO)2 POCl/pyridine; (e) 1, H2/PtO 2 2, H + ; (f) HOCH2CHO/FDP aldolase, then acid phosphatase.

Synthesis of[2,3-13C]Dihydroxyacetone Phosphate and[2,3J3C]Xylulose. To a suspension of sodium hydride (480 mg, 20 mmol) in 60 ml of dry T H F is added 1 ml of benzyl alcohol, and the suspension is stirred for 30 min at room temperature (Fig. 9). One gram (7.1 mmol) ofbromo[1,2-~3C]acetic acid and 100 mg of tetra-n-butylammonium iodide are then added, and the resulting solution is stirred for 72 hr at room temperature. The mixture is evaporated to give a solid residue, which is dissolved in 30 ml of water and extracted with dichloromethane (3 × 15 ml) to remove excess benzyl alcohol. The water layer is acidified with 1 N HCI and then extracted with dichloromethane (3 x 20 ml). The combined organic layers are dried over anhydrous sodium sulfate. Evaporation of the solvent gives 0.906 g of [1,2-13C]benzyloxyacetic acid. lH NMR (CDC13/TMS) 8 4.15 [2H, dd, J = 4.5 Hz (H-13CI) and 144 Hz (HJ3C2), J3CH2-], 4.65 [2H, d, J = 4.2 Hz (H-laC2, -CH20-)], 7.36 (5H, s, phenyl), 10.5 (1H, bs, COOH). 13C NMR 8 66.54 (13C2, d, J = 61.0 Hz), 175.71 (laCl, d, J = 61.0 Hz). The crude product is treated with diazomethane in ether. The ether is removed to give an oil residue which is purified by passing through a short silica gel column (CH2C1z) to give 0.983 g (76% yield) of a colorless oil. lH NMR (CDCI3/TMS) 8 3.76 [3H, d, J = 3.6 Hz (H-13C1), -OCH3], 4.11 [2H, dd, J = 4.6 Hz (H-13C1) and 144 Hz (H-13C2), -OI3CH/], 4.63 [2H, d, J = 4.4 Hz (H-13C2), -CH20-], 7.35 (5H, s, phenyl), laC NMR 6 67.07 (13C2, d, J = 63.0 Hz), 170.79 (13C1, d, J = 63.0 Hz). To a solution of 1.0 g (8.3 mmol) of 1,3-dithiane in 30 ml of dry T H F

610

ENZYMES

[26]

is slowly added 4 ml of n-BuLi (6.4 mmol) at - 4 0 °. The resulting clear pink solution is continuously stirred for 3 hr at - 2 5 °. This solution is transferred under N 2 to the above compound (5.4 mmol) dissolved in 10 ml of dry T H F at - 78 °. The temperature is raised to - 20°, and the mixture is stirred for 3 hr. T H F is removed in v a c u o . The residue is dissolved in dichloromethane and washed successively with water and brine and then dried over anhydrous sodium sulfate. The solvent is removed in v a c u o to give an orange-colored crude product which is purified by silica gel column chromatography to give 1.35 g (93%) of 1 (Fig. 9). 1H NMR (CDC13/TMS) 8 1.9-2.2, 2.45-2.6, and 3.2-3.4 (6H, m, -SCH2CH2CH2S-), 4.28 [2H, dd, J = 3.8 Hz (H-ISC1) and 143 Hz (HJSC2), -O13CH2-],4.48 [1H, d, J = 4.0 Hz (H-lSC1), -SCHS-], 4.60 [2H, d, J = 4.0 Hz (H-13C2), -CH20-], 7.35 (5H, s, phenyl). PC NMR 8 72.26 (_CH2-CO, d, J = 45.5 Hz), 200.48 (CO, d, J = 45.5 Hz). A mixture of 1 (1.35 g, 5.0 mmol), trimethyl orthoformate (8 ml), p-toluenesulfonic acid (135 mg), and dry methanol (20 ml) is heated at reflux in a flask equipped with a Dean-Stark water separator. After 20 hr, the reaction is cooled, and 10 ml of saturated sodium bicarbonate is added. The resulting solution is stirred for 30 min at room temperature and then extracted with CH2C12. The organic layer is washed with brine and dried over anhydrous sodium sulfate. Removal of the solvent gives a crude product, which is dissolved in 50 ml of acetonitrile-water (4 : 1) containing 1.5 g of CaCO 3 and 5 ml of methyl iodide. The resulting mixture is stirred for 6 hr at 65 °. Removal of the acetonitrile and excess methyl iodide gives a slurry, which is extracted with dichloromethane. The combined extracts are washed with water and brine, then dried over anhydrous sodium sulfate. Removal of the solvent gives a residue, which is chromatographed over an aluminum oxide column (neutral). Elution with n-hexane-ethyl acetate (4 : 1) gives 365 mg (51%) of 2 (Fig. 9) and 213 mg of unreacted substrate. The recovered substrate is repeatedly treated as above to give another 102 mg of 2 (total yield 65%). IH NMR (CDCls/TMS) 8 2.25 (1H, bs, OH), 3.27 (6H, d, J = 3.6 Hz, -OCHs), 3.54 (2H, dd, J = 3.8 and 143 Hz, -OCH2-), 3.69 (2H, m, -CH20-), 4.58 (2H, d, J = 4.4 Hz, -OCH20-), 7.33 (5H, s, phenyl). 13C NMR 8 68.32 (OCH2, d, J = 52.5 Hz), 99.98 [C(OMe) z, d, J = 52.5 Hz]. To 340 mg of 2 in 5 ml of dry pyridine cooled in ice water is added dropwise 0.371 ml (1.8 mmol) of diphenyl phosphorochloridate over a period of 5 min. The reaction vessel is then stoppered and left overnight at room temperature. A few drops of water are added to destroy the excess phosphorylating reagent. Thirty milliliters of benzene is added, and the resulting solution is washed successively with 15 ml each of water, saturated sodium bicarbonate, and brine. The benzene layer is dried over

[26]

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

611

sodium sulfate and concentrated to a syrup, which is purified by silica gel column chromatography to give 626 mg (91%) of 3 (Fig. 9). 1H NMR (CDCI3/TMS) 8 3.20 (6H, d, J = 3.8 Hz, -OCH3), 3.45 [2H, dd, J = 4.0 and 144 Hz (-OCH2-)], 4.30 (2H, m, -CH2OP-), 4.46 (2H, d, J = 4.2 Hz, -OCH20-), 7.1-7.4 (15H, m, 3 phenyl). 13C NMR 8 65.78 (OCH2, d, J = 53.0 Hz), 99.52 [C(OMe)~, dd, J = 53.0 and 10.0 Hz, C-P]. The two phenyl groups and the benzyl group of 3 are removed by hydrogenation at atmospheric pressure in 15 ml of methanol containing 63 mg of platinum oxide. After 5 hr, the catalyst is removed by filtration and washed with methanol. The filtrate is concentrated under reduced pressure to a syrup, which is used directly for the next step. IH NMR (D20/CH3OH) 8 3.31 (6H, d, J = 3.7 Hz, -OCH3), 3.64 (2H, dd, J = 3.32 and 142 Hz, -OCH2-), 3.91 (2H, m, -CH2OP-). 13C NMR 8 57.38 (OCH 2, d, J = 51.5 Hz), 100.96 [C(OMe)2, dd, J = 51.5 and 11.0 Hz, C-P]. To remove the dimethoxy group, this compound is dissolved in 4 ml of 1 N HCI and stirred for 4 hr at 40°. Then the reaction solution is evaporated under reduced pressure to remove methanol, and the pH is adjusted to 6.8 with 1 N NaOH. The crude product 4 (Fig. 9) is used directly for the aldol condensation. Hydrate form: ~H NMR (D20/CH3OH) 8 3.60 [2H, dd, J = 3.14 Hz (H-13C2) and 142 Hz (H-13C3), -OCH2-], 3.93 (2H, m, -CH2OP-). 13C NMR (D20/CH3OH) 8 63.51 (13C3, d, J = 48.5 Hz), 94.21 [13C2, dd, J = 48.5 Hz (13C-13C) and 9.5 Hz (13C-p]. Keto form: 1H NMR (D20/CH3OH) 8 4.49 [2H, dd, J = 3.54 Hz (H-13C2) and 144 Hz (H-13C3), -OI3CH2-], 4.71 [2H, dd, J = 3.88 Hz (H-13C2) and 8.28 Hz (H-P), -CH2OP-]. 13C NMR 8 65.16 (13C3, d, J = 42.5 Hz), 208.23 [13C2, dd, J = 42.5 Hz (13C-13C)and 7.0 Hz(13C-p)]. To the above solution, 162 mg (2.7 mmol) of glycoaldehyde and 22 units of rabbit muscle FDP aldolase are added, and the mixture is stirred for 14 hr at room temperature. Acid phosphatase (5 mg) is added, and the resulting solution is stirred for an additional 6 hr. The solution is lyophilized to give a residue, which is purified by silica gel column chromatography (dichloromethane-methanol 5 : 1, v/v) to give 102 mg of D-xylulose. The ratio of the three isomers (Sa: 5b : 5¢; Fig. 9) is 1 : 4 : 1.3 based on the 13C NMR spectroscopy analysis. 13C NMR (CD3OD/CH3OH) for 5a8 82.41 [13C3, d, J = 46.5 Hz (13C-13C)], 107.03 (13C2, d, J = 46.5 Hz (13C-13C). 5b 8 78.05 [13C3, d, J = 43.6 Hz (13CJ3C)], 104.47 [13C2, d, J = 43.6 Hz (13C-13C)]. 5C ~ 76.86 [13C3, d, J = 43.5 Hz (13C-13C)], 213.95 [13C2, d, J = 43.5 Hz (13C-13C)].

Sialic Acid Aldolase-Catalyzed Synthesis of N-Acetyl-D-neuraminic Acid (Neu5Ac, Sialic Acid). To a solution of 9.6 g (40 mmol) of N-acetylD-mannosamine, 44.9 g (400 mmol) of sodium pyruvate, and 0.12 g (1 mmol) of NaH2PO4 in 260 ml of water (pH 7) is added 29 U of insoluble

612

Er~ZVMES

[26]

PAN-immobilized 55 Nacetylneuraminic acid aldolase (EC 4.1.3.3, N-acetylneuraminate lyase, from Toyobo, New York). The reaction is followed by measuring the time-dependent changes in the 1H NMR signals of the N-acetyl groups in the starting material and product. 56 After 10 days the reaction has reached 91% completion, and the enzyme gels are removed by centrifugation. Fifty-two-milliliter portions of the solution are passed through a 2.5 × 40 cm column of Dowex 50-X8 (H ÷) with water elution. The portion of the eluant which contains the product is lyophilized to a paste. The paste is slurried in ethyl acetate, and the Neu5Ac product recovered by filtration. The collected white powder is dried under a stream of dry air to yield 2.1 g (85%) of Neu5Ac as a monohydrate. The ~H NMR spectrum of the product obtained is identical to the reported spectrum) 7 The product is crystallized from water-acetic acid (1 : I0, v/v) [rap 186°-188 °, literature 187°-189 ° (Ref. 58), 184°-186 ° (Ref. 59)]. Methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-2-deoxy-[3-D-neuraminate.

A

300-mg sample of the glycosyl chloride (see Fig. 10), prepared as previously referenced from 200 mg of Neu5Ac, 6° is dissolved in 40 ml of toluene. After the solvent is distilled to half-volume, 0.4 ml (5 equivalents) of triethylamine is added to the hot solution. Heating is resumed and the reaction monitored by TLC (silica gel, 2% ethanol in ethyl acetate) until complete consumption of the glycosyl chloride is observed (20-30 min). The solution is evaporated in vacuo, and the residue is chromatographed on a 21 × 150 mm flash column using 2% methanol in chloroform as the eluant. The product fractions are pooled, evaporated in vacuo, and further azeotroped with toluene to remove any residual chloroform. The residue is dissolved in 30 ml of 7 : 3 : 1 toluene-ethyl acetate-methanol. The solution is transferred to a low-pressure Parr bottle and hydrogenated over 200 mg of 10% palladium-on-carbon under 40 psi of hydrogen gas. After 20 hr the excess hydrogen gas is released, the solution filtered, and the catalyst washed successively with I0 ml of ethyl acetate and 10 ml of methanol. The combined filtrate and washings are evaporated to dryness. The residue is taken up in 20 ml of ethyl acetate and washed 55 A. Pollak, H. Blumenfeld, M. Wax, R. L. Baugh, and G. M. Whitesides, J. Am. Chem. Soc. 102, 6324 (1980). 56 M.-J. Kim, W. J. Hennen, H. M. Sweers, and C.-H. Wong, J. Am. Chem. Soc. 110, 6481 (1988). 57 E. B. Brown, W. S. Brey, Jr., and W. Weltner, Jr., Biochim. Biophys. Acta 399, 124 (1975). 58 j. E. Martin, S. W. Tanenbaum, and M. Flashner, Carbohydr. Res. 56, 423 (1977). 59 M. F. Czarnieck and E. R. Thorton, J. Am. Chem. Soc. 99, 8273 (1977). 6o H. Ogura, K. Furuhata, M. Itoh, and Y. Shitori, Carbohydr. Res. 158, 37 (1986).

[26]

ENZYMATIC CATALYSISIN ORGANICSYNTHESIS

MeOH Dowex 50 (H')~

Neu5Ac

HO ~ HO

AcNH~

O

613

O~ CO2Me

I AcCl AcO A_ ~

AcO ~O~.sCO2M

Et3N, ~cOA ~ - @ ~ " ~

H2/Pd-C EIsN

H2/Pd-C AcO AcOA c O ~ " ~ ~

AcO

A c N H ~

O-~C02Me

52 % from Neu5Ac

O~CO2 M

AcNHAcO-~-'7~,/

AcNHAcO--~/-~'~7/

CO2Me

,co

AcNHAcO~-/~O'7~'~/ 50% from Neu5Ac

FIG. 10. Synthesis of a and fl isomers of 2-deoxysialic acid from sialic acid prepared enzymatically.

successively with 10-ml portions of 10% aqueous potassium hydrogen sulfate, water, saturated aqueous sodium bicarbonate, and brine. After being dried over magnesium sulfate, the solution is evaporated in v a c u o . The residue is dissolved in chloroform and loaded into a 2 × 360 mm silica gel column. The product is eluted from the column with 600 ml of 1 : 1 ethyl acetate-hexane followed by 0-2% ethanol in ethyl acetate. The fractions with product are pooled and evaporated to yield 150 mg (52%) of the title compound. The product is crystallized from dichloromethane-hexane (mp 181°-183°). lH NMR (CDC13) 6 1.82 (ddd, 1 H, J --- 12.9, 12.2, 11.2 Hz, H-3a), 1.86 (s, 3-H, N-Ac), 2.01 (s, 6-H, 2-OAc), 2.04 (s, 3-H, OAc), 2.11 (s, 3-H, OAc), 2.38 (ddd, 1 H, H-3e, J = 12.9, 4.9, 2.3 Hz), 3.68 (dd, 1 H, J = 10.4, 2.2 Hz, H-6), 3.74 (s, 3 H, OMe), 3.99 (ddd, 1 H, J = 10.4, 10.3, 10.0 Hz, H-5), 4.04 (dd, 1 H, J = 2.3, 12.2 Hz, H-2), 4.12 (dd, 1 H, J = 7.4, 12.4 Hz, H-9), 4.64 (dd, 1 H, J = 2.5, 12.4 Hz, H-9'), 5.01 (ddd, 1 H , J = 4.9, 10.3, 11.2 Hz, H-4), 5.21 (ddd, 1 H , J = 2.5, 4.7, 7.4, H-8), 5.34 (dd, 1 H, J = 2.2, 4.7 Hz, H-7), 5.56 (d, 1H, J = 10.0 Hz,

614

ENZYMES

[26]

NH), 13C NMR (CDCI3) 8 20.79, 20.81, 20.89, 20.93, 23.22, 33.49, 49.67, 52.49, 62.43, 68.13, 71.34, 71.61, 74.49, 77.65, 168.99, 170.21, 170.31, 170.38, 170.57, 170.98. LRMS (m/z) 475,432,416, 373,356, 355,330, 313, 300, 295, 253, 186, 101. HRMS (m/z) M + 1 calculated for C20Ha0NOl2 476.17680, observed 476.17510; M calculated for C20HE9NOI2475.16898, observed 475.16588. N-Acetyl-2-deoxy-fl-D-neuraminic Acid. A rapidly stirred suspension of 110 mg (0.23 mmol) of methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-2-deoxyfl-o-neuraminate in 10 ml of water is adjusted to pH 11.5 and maintained at pH 11.5 by the addition of 0.25 N NaOH. After 1.5 hr, 5 equivalents of hydroxide has been consumed, and no further drop in pH is observed. The solution is passed through a 1.0 × 22 cm column of Dowex 50-X8 (H + form) and lyophilized to yield 70 mg (-100%) of 2-deoxy-/3-o-Neu5Ac as a hygroscopic white powder. The powder is crystallized from methanol-diethyl ether Imp 2270-228° (dec.)]. IH NMR (D20) 8 1.66 (ddd, 1H, J = 12.8, 12.0, 10.8 Hz, H-3a), 2.04 (s, 3H, NAc), 2.43 (ddd, 1H, J = 12.8, 4.4, 2.4 Hz, H-3e), 3.50 (dd, 1H, J = 9.2, 1.1 Hz), 3.59-3.66 (m, 2H), 3.79-3.92 (m, 4H), 4.27 (dd, 1H, J = 12.0, 2.4 Hz, H-2). 13C NMR (D20) ~ 22.95, 36.88, 52.91, 64.04, 69.19, 70.67, 71.10, 74.47, 76.58, 175.68, 175.75. Fast atom bombardment (FAB) MS (re~e) 294 (M + 1), 276, 185, 93. Analysis, calculated for C11H19NO8 C 45.06, H 6.53; found C 45.20, H 6.41. N-Acetyl-2-deoxy-a-D-neuraminic Acid. Methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-2-deoxy-ot-o-neuraminate54is similarly deprotected in quantitative yield to give 2-deoxy-c~-D-Neu5Ac (mp 184°-185°). 1H NMR (D20) 8 1.92 (ddd, 1H, J = 13.6, 11.6, 6.6 Hz, H-3a), 2.04 (s, 3H, NAc), 2.54 (ddd, IH, J = 13.6, 4.8, 1.6 Hz, H-3e), 3.53 (dd, 1H, J = 9.2, 1.2 Hz), 3.59-3.66 (m, 1H), 3.70-3.78 (m, 1H), 3.78-3.87 (m, 4H), 4.71 (dd, 1H, J = 6.6, 1.6 Hz, H-2). 13C NMR (O20) 8 22.80, 34.26, 53.16, 63.89, 68.44, 69.08, 71.95, 72.91, 74.46, 176.06, 176.19. Analysis, calculated for CI1HIaNO8 C 45.06, H 6.53; found C 44.83, H 6.37. The anomeric configuration is established by comparison with the a isomer. 56 The proton DQCOSY spectrum is shown in Fig. 11. The offdiagonal elements, indicating coupling to H-3a and H-3e, clearly show that the absorption at 4.04 ppm is due to H-2. The larger H-2 coupling constants found in this isomer and the deprotected product compared to the ct isomers confirm the /3 configuration. The proton coupling pattern also indicates the 2C5 ring conformation.

Construction of Fab Libraries from Immunized Mice Amplification of Fab Genes Corresponding to Heavy and Light Chains from Immunized Mice. The spleens of mice immunized with a transition

[26]

615

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

AcO AcO

Jb

F~. (PPM)

COOMe

J •-

A c N H A--~-O/

G :NH

i

=..~ . . . . . . . . . .

H-7 WH-8

,:, ,'

5.0

".° ,

H-4

°T"

I H-9 '

4.5

i" •....: .....

~? i ,

;,

___

~n - 9 . _ ~ n _

4.0

2

n-S

',' la "----IS

~-6

c~

3.5

3.0 %

2.5

~- - ::__,

"-~

i

2.0

2(3Ac

I

5.5

/

5:o

4:5

4:o

a:s

3:o

2:5

2:o

F2 (PPM) FIG. I1. Proton phase-sensitive double-quantum filtered homonuclear correlated spectroscopy (DQCOSY) of methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-2-deoxy-fl-D-neuraminate (6).

state analog are used to extract total RNA according to the method described by Chomczynski and Sacchi. 61 The isolated RNAs are used for cDNA synthesis using oligo(dT) as primer (Riboclone cDNA synthesis system from Promega, Madison, WI). In a typical 25-/zl transcription reaction, 5/xg of RNA is annealed with 500 ng of the oligo(dT)~0 at 70° for 5 min. Subsequently, the mixture is adjusted to contain 1 mM of dinucleotide triphosphates (dNTPs), 50 mM Tris-HC1, pH 8.3, 75 mM KC1, 10 mM MgC12, 0.5 mM spermidine, 10 mM dithiothreitol (DTT), 4 mM sodium pyrophosphate, 1 unit RNasin ribonuclease inhibitor, and 10-15 units avian myeloblastosis virus (AMV) reverse transcriptase. The volume is then brought up to 50/xl by the addition of diethyl pyrocarbonate (DEPC)61 p. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987).

616

ENZYMES

[26]

Sac I Xba I 5 3t~L ~ S t- 1 R 13'15 Lc 1 cloningvectorpreparedfromAZAPII 1) Xba I 2) Sac I

,,,,,,

0-.o o

OH

S

oP

PO__

IIIIII

COS end

1) Annealing 2) Dialysis 3) Extraction,precipitation

°"3',,

5'PO 3' 1) 2)

(light chain insert) 3) Ligation I

i

oP

I

,

1) in vitropackaging 2) Infectionof XLI Blue E coli

FIG. 12. Preparation of cloning arms for the construction of catalytic Fat) fragments. treated water. PCR amplification is performed in a 100-/zl reaction mixture containing 3/xl o f the reverse transcription product, 400 nmol of 3' variable heavy chain (VH) or variable light chain (VL) primer and one of the 5' V H or V L primers, 42 200/xM of different dNTPs, 50 m M KC1, 10 m M TrisHCI (pH 8.3), 20 m M MgCI2, 0.01% gelatin, 0.1% Triton X-100, and 2 units o f Thermus aquaticus D N A polymerase. The reaction is overlaid with mineral oil and subjected to 35-40 cycles of amplification. The cycle conditions are adjusted according to the primer characteristics. Typically, denaturation at 92 ° for 1 min, annealing at 52 ° for 2 min, and elongation at 72 ° for 1.5 min will be used.

Preparation of Bacteriophage h Arms from Light Chain (Lcl) and Heavy Chain (Hc2) Vectors. L c l or Hc2 vector D N A (see Fig. 12) prepared from h ZAP 42 is suspended in a p r o p e r buffer provided by Boehringer Mannheim Biochemical Co. (Indianapolis, IN) ( L c l in A buffer and Hc2

[26]

ENZYMATIC CATALYSIS IN ORGANIC SYNTHESIS

617

T A B L E II EFFICIENCY OF L c l ARMS PREPARED BY DIFFERENT PROCEDURESa

Procedure Digestion with restriction e n z y m e s Digestion and dephosphorylation Digestion, annealing, and dephosphorylation Digestion, annealing, dialysis, and dephosphorylation

A r m s efficiency (pfu//zg D N A ) 1 × 105

Background (pfu//xg DNA)

0 2.9 x 106

2 - 4 x l0 4 1.2 x 104 5 × 105

9.5 × 106

2.1 × 105

Efficiency and b a c k g r o u n d were determined b a s e d on plaque-forming units (pfu).

in H buffer) and digested with two restriction enzymes, one in each step (Lcl is digested with XbaI and SacI, and Hc2 is digested with XhoI and SpeD, at a concentration of 3 units//zg vector DNA (1 unit will completely cleave 1/zg of X DNA in 1 hr at 37° in a total volume of 25/xl incubation buffer). After incubation at 37 ° for 30 min, MgCI2 is added to reach a final concentration of 10 raM, and the solution is further incubated with 42 ° for 1 hr. The digested DNA solution is then dialyzed against a 2000-fold volume of Tris-EDTA (TE) buffer (10 raM-1 rnM, pH 7.5) for at least 2 hr. After dialysis, the DNA solution is extracted with phenol-chloroform, followed by addition of ethanol (70% final concentration) to precipitate DNA. The DNA pellet is vacuum-dried in a Speed-Vac and resuspended in Tris-acetate (TA, 33 mM Tris, pH 7.8, 0.5 mM dithiothreitol, 100/zg/ ml BSA, 5 mM CaC12, 10 mM Mg-acetate, 66 mM K-acetate.) buffer (Epicentre Technologies, Madison, WI). Dephosphorylation is carried out as described in the protocol of Epicentre Technologies by using HK phosphatase. The dephosphorylated DNA is further extracted with phenol/chloroform and precipitated with 70% ethanol. The precipitated arms are dissolved in TE buffer (pH 7.5) at a concentration of 0.5-1/zg/ml. The three steps involving annealing, dialysis, and dephosphorylation improve the efficiency of the cloning arms by a factor of about 100 as compared with previously reported procedure42 (Table II). Construction of Heavy and Light Chain Libraries. The DNAs obtained from the PCR amplification are purified on 0.6% agarose gels. The DNA band corresponding to about 600 base pairs (bp) is separated from the agarose gel and electroeluted. The DNAs are then extracted with phenol-chloroform and precipitated with ethanol overnight at - 2 0 °. The precipitated DNAs are dissolved in a proper restriction enzyme buffer supplied by Boehringer Mannheim (H buffer for heavy chains and A buffer for light chains) and digested with the corresponding restriction enzymes (70 units//zg DNA, heavy chain DNA by SpeI and XhoI and light chain

618

ENZYMES

[26]

Heavy Chain cDNA Library

Light Chain eDNA Library

Digested with Xhol

I andspe Light Chain Vector Arms

SacI

I

I.,ightChain

[ XbaI

XhoI

[

net,, Ou~

[

SpeI

IBacteriophage T4 DNA ligase

Heavy Chain Vector Arms

I Bacteriophage ~ T4 DNA ligase !

Package in vitro into bacteriophage k and plate on XLI-Blue E. coil plates

q

I Light Chair~ Library |

~,

Heavy Chain Library

DNA purification, digested with Mlu I, ] dephosphorylated at 5' en._...dand cleaved with EcoR I

DNA purification, digested with Hind III, dephosphorylated at 5'end and cleaved with EcoR I

-1~ Random combination

FIG. 13. Construction of recombinatorial Fab libraries.

DNA by XbaI and SacI; all restriction enzymes are from Boehringer Mannheim) for 2 hr (see Fig. 13). The digested DNAs are then recovered by phenol-chloroform extraction and ethanol precipitation and resuspend in TE buffer. The DNAs are used as inserts, ligated to the cloning arms, and packaged with a packaging kit (Stratagene Co., San Diego, CA). The size of the library is estimated by the total plaques formed, which are identified as positive Fab expression plaques by an enzyme-linked immunosorbent assay (ELISA) method. 42 The packaged phages are then amplified in LB agar plates (15 cm diameter) and eluted with SM solution. The libraries are stored in 7% dimethyl sulfoxide at - 70 ° or 0.5% (v/v) chloroform at 4 ° . Combination of Heavy Chain and Light Chain Libraries. The heavy and light chain libraries are amplified in a 2-1 LB medium, and the DNA

[26]

ENZYMATIC CATALYSISIN ORGANIC SYNTHESIS

,

i ReactantsI Enzyme(s) ~

1. IEnzymeknown ? I l yes

619

I Products I

no

2. I Ad~taateActivity71

no

" "

tic I e °r72t T°st I' "I Catal An"ZY [

t/v:t or

~ yes 3. ] Adequate Stability ? ]

~yes 4. I ReadilyAvailable ? I

~yes

o~f.-~/,~..

['~"~,~.~Matagenesis I Molecular Modeling I

5. I ProcessDevelopment I FIG. 14. Strategyfor the developmentof enzymaticcatalysts.

of the light chain library is digested with MluI, dephosphorylated at the resulting 5' end, and then further digested with EcoRI. On the other hand, the DNA of heavy chain library is digested with HindlII, dephosphorylated, and then cleaved with EcoRI. The generated DNAs are then ligated at the EcoRI site and packaged by a packaging kit. The expression of Fab is detected by ELISA. Amplification of the Fab combinatorial library is carried out as that described for the heavy and light chain library construction, and the combinatorial libraries are preserved at 4° with 0.5% chloroform or at - 7 0 ° with 7% dimethyl sulfoxide. To screen for antigen-binding Fab's, a standard plaque lift method 42 is used. Protein expression is induced with isopropyl-/3-D-thiogalactopyranoside (IPTG), and the expression library is screened against ~zSI-labeled bovine serum albumin (BSA) conjugated with the antigen. The positive plaques are then excised with helper phage M13 mp8 and infected to FIMC 1060 for isolation of desired Fab's. Such Fab's can be further examined for possible catalytic activities.

Conclusion and Perspectives Enzymes have a proven record as useful catalysts for organic synthesis, particularly for the synthesis of chiral intermediates and biologically active substances. Synthesis based on a combined chemical and enzymatic approach, called chemoenzymatic synthesis, will become an important

620

ENZYMES

[27]

and effective strategy in synthetic organic chemistry. New transformations based on known or new enzymes will continue to be exploited. It is worth noting that the number of enzymes reported so far (about 2300) represents only approximately 2% of the total number of enzymes existing in nature. Many unknown enzymes remain to be explored. Known enzymes or proteins can be altered through site-directed mutagenesis or chemical or biological modification to provide new catalytic activities. New protein catalysts specific for a predetermined reaction are now available through the catalytic antibody approach. Multiple enzyme systems required for efficient synthesis of complex molecules such as antibiotics may be constructed within a cell via genetic manipulation and metabolic pathway engineering. It seems fair to say that virtually all kinds of protein catalysts can be constructed from the 20 common amino acids. Figure 14 illustrates our strategy for the development of a specific and efficient protein catalyst for the transformation of a desired reaction. Acknowledgments This work was supported by the National Institutes of Health (GM44154). We thank Professor Richard Lerner for advice on the work involvingFab libraries.

[2 7] M o d i f i c a t i o n o f E n z y m e C a t a l y s i s b y E n g i n e e r i n g Surface Charge

By GREGORIO ALVARO and ALAN J. RUSSELL Introduction The rational modification of enzyme catalysis has been an elusive goal of biochemists for many years.l Until the combination of the techniques of recombinant DNA methodologies and DNA sequencing enabled "protein engineers" to introduce specific changes in the amino acid sequence of proteins via site-directed mutagenesis,2 the alteration of enzyme characteristics was limited to chemical modification? Chemical alterations were rarely specific and often resulted in significant changes in protein structure. Ulmer's insightful review I in 1983 described the properties of enzymes which could be altered in order to "improve" biocatalyst properties, and l K . M. U l m e r , Science 219, 666 (1983).

2 M. J. Zollerand M. Smith,D N A 3, 479 (1984). 3A. Gounaris and M. Ottensen, C. R. Tray. Lab. Carlsberg 35, 37 (1965). METHODS IN ENZYMOLOGY, VOL. 202

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.