Structural studies and synthetic applications of Baeyer-Villiger monooxygenases

35 f OCUS

Structural studies and synthetic applications of Baeyer-Villiger monooxygenases Andrew Willetts Baeyer-Villiger

monooxygenases

1BVMOs) are enzymes able to perform highly

regio- plus stereoselective nucleophilic and electrophilic biooxygenations on various substrates. The resultant chiral products (lactones and sulfoxides) can be valuable for the chemoenzymatic

synthesis of a wide range of useful compounds. Recent

studies have provided a number of alternative active-site models that attempt to explain the exquisite and unusual selectivity of BVMOs. This article reviews some of the established applications,

and considers the merits of the various predictive

models. The Baeyer-Villiger monooxygrnases (BVMOs) (EC I .13.13.x) are an interesting group of flavoproteins with considerable proven ability for yielding key chiral products of value in the chemoenzymatic synthesis of a wide range of useful compounds’. These enzymes exhibit the rare characteristic, shared with some other notable biocatalysts such as ribulose bisphosphate carbox$ase/oxygenase, of being able to catalyse two mechanistically different types of biochemical reactions, apparently within the confines of the same active site. BVMOs were first recognized over 40 years ago. during the mltlal era of steroid biotransformations, as carbonyl monooxygenases (Fig. la) with the ability to catalyse the nucleophilic oxygenation of ketones and aldehydes in a manner analogous to the established peracid-catalysed organic chemical reaction from which they take their name’. However, more recently it has been recognized that these enzymes can also catalyse the electrophilic oxygenation of various heteroatoms, as illustrated by their ability to form sulfoxides from organosulfides’. Both types of reaction can proceed with exquisite selectivity. For example, cyclohexanone monooxygenase (CHMO) purified from cyclohexanol-grown A&zctobarrer &oacetii-us (NCIMB 9871) yields homochiral (S)-5-methyl-(E)-lactone [enantiomeric excess (e.e.) = 98’S] from +methylcyclohexanones, and (R)-methyl phenyl sulfoxide (e.e. = 99%) from the equivalent sulfide”. BVMOs in nature Almost without exception, the enzymes date have been found in microorganisms,

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0167

studied to including

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bacteria

of the genera Ai-irletobnrter, P~e-ecrdomorlas, XanR~U&ON-US and ~Ymdia, and fungi of the genera Crrrddria, Dresdllcva, Ea$iilia, Cunu2ir@ilrnel/a and Cylindroiav~ow. In bacteria, the natural role of these enzymes appears to be to catalyse one or more key steps in various oxidative catabolic pathways:. whereas in fungi they appear to play some, as yet, poorly characterized role in the programme of biochemical events that accompanies the switch from primary to secondary metabolism. When, as in the case of cyclohexanol-grown A. doacetirr~s, there is only one induced BVMO present in the microbial cells, it is possible to use various washed-cell preparations of the biocatalyst to perform biotransformations. However, the presence of an active lactone hydrolase in the same catabolic pathway can result in low yields of lactone products unless a suitable inhibitor, such as tetraethylpyrophosphate, is added to the reaction mixturex. In bacteria such ‘1s camphor-grown Psetrdomonas p~rtida, which contains various induced BVMOs with contlicting selectivities. the use ofwhole-cell preparations is impractical”. Both problems can be eliminated by using purified enzyme preparations. One potential problem of using pure enzymes, namely the cost of using stoichiometric amounts of the relevant reduced nicotinamide nucleotide (Table l), has been resolved by the successful use of appropriate linked-enzyme coenzyme-recycle systetns (NADP/H + glucose dehydrogenase. NAD/H + formate dehydrogenase). The coenzyme recycling system for each BVMO has been developed as a continuously-operated process using enzyme membrane reactor-based technologv’“.” Bv choosing the appropriate substrate and a CI 1 & suitable enzyme complement, it is possible to operate a clozed-loop recycling procedure that eliminates the need for a sacrificial substrate (Fig. 1b). thhai-rcr,

PII. SO167 7799(97)01004-4

TIBTECH FEBRUARY1997WOL15)

0Testololactone

Progesterone

b

HO

DH

Figure 1 (a) Characterized pathway of progesterone metabolism in Cylindrocarpon radlcicola2, containing two examples (denoted by l ) of BaeyerVilliger monooxygenase catalysed reactions. (b) lntrasequential regeneration of nicotinamide nucleotide coenzymes using a coupled alcohol dehydrogenase (DH) and Baeyer-Villiger monooxygenase (MO112 Structural

features

of BVMOs

The best characterized BVMO to date is cyclohexanone monooxygenase from A. calconcrticus. The enzyme is monomeric with a molecular mass of 39 kDa: although a complete three-dimensional structure remains to be established(seebelow), a comprehensive seriesof studiesby Trudgill’s group has concluded that the enzyme has only one active site”. Some characteristicsof CHMO and a number of other BVMOs that have been purified are comparedin Table 1. Although the complete sequenceis only available for CHMO (Ref. 14), partial N-terminal sequences have been obtained from a sufficient number of these flavoproteins (Box 1) to justify dividing them into two distinct types, known asType 1 and Type 2 enzymes (G. Grogan. PhD Thesis, University of Exeter, 1995). Type 1 enzymes are FAD binding and NADPHdependent, and both coenzymes plus the substrateall bind to the samepolypeptide subunit: some of these enzymes are monomeric, whereas others have a homo-oligomeric quaternary structure. By contrast, Type 2 enzymes are FMN binding and NADHdependent and consistof two distinct types of subunits, one of which binds the carbonyl substrate and the other the nicotinamide nucleotide, effectively serving asan NADH dehydrogenase. A notable feature of all the Type 1 enzymes is the occurrence, as observed in a number of other TIBTECH FEBRUARY1997WOL151

FAD-binding enzymes, of the characteristic GxGxxG consensusnucleotide sequence located near to the N-terminus, which representsthe binding site for the adenosine moiety of FAD (Ref. 15). Although it remains to be confirmed by further examples, it appearsthat a distinct feature of Type 1 BVMOs that distinguishes them from other FAD-dependent enzymes is the presence of an invariant aspartic acid residue immediately upstreamfrom the FAD-binding site. The functional significance, if any, of this residue remainsto be established.The N-terminal sequences of 2,5-diketocamphane 1,2-monooxygenase (2,5DKCMO) and 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) [two Type 2 FMN-binding and NADH-dependent isozymes responsiblefor the degradation of (+)- and (-)-camphor, respectively, in Pseudomona putida’ are clearly different from Type 1 enzymes but show considerable homology to each other aswell asto various bacterial luciferases,which act asBVMOs in performing the FMN and NADHdependent oxygenation of dodecanal to dodecanoic acid”. Enantiodivergent biotransformations synthetic applications

including

The selectivities of some of the nucleophilic oxygenations performed by this group of enzymes are as unusualasthey areimpressive.In somecases,particular

57 I- ecus

Table 1. Characteristics

of some microbial Baeyer-Villiger

monooxygenases

Enzyme and source microorganism

Coenzyme

No. of proteins

Subunit composition

Cyclohexanone monooxygenase Acinetobacter calcoaceticus NUMB 9871 Cyclohexanone monooxygenase Nocardia globerula CL1 Cyclohexanone monooxygenase Xanthobacter sp. Cyclopentanone monooxygenase Pseudomonas sp. NCIMB 9872 Tridecan-2-one monooxygenase Pseudomonas cepacia Diketocamphane monooxygenase Pseudomonas putida NCIMB 10007 2oxo-As-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase Pseudomonas putida NCIMB 10007 Steroid monooxygenase Cylindrocarpon

NADPH

1

Singlepolypeptide chain

NADPH

1

NADPH

1

NADPH

1

Singlepolypeptide chain Singlepolypeptide chain 3-4 identicalsubunits

NADPH

1

Two identicalsubunits

NADH

2

NADPH

Molecular mass (kD4

Mole coenzyme/ mole protein

PH optimum

59

1 FAD

9.0

53

1 FAD

8.4

50

1 FMN

8.8

200

1 FAD

7.7

123

1 FAD

7.8-8.0

1 FMN

9.0

1

(0 Two identicalsubunits 78 (ii) Onepolypeptidechain 36 Two identicalsubunits 106

1 FAD

9.0

NADPH

1

Two identicalsubunits

155

1 FAD

7.8

NADH

2

Ii) Two identicalsubunits 70 (ii) Onepolypeptidechain 30

1 FMN

8.5-9.0

radicicola ATCC 11011

Oxocineolemonooxygenase Pseudomonas

flava UQM 1742

Box 1. Sequence data for Types 1 and 2 Baeyer-Villiger

monooxygenases

Type 1: FAD- and NADPH-dependent Binding

motif for N-term....

adenosine moiety of FAD .5-25 amino acids................G,x._G.x.x.E..........................

l Cyclopentanone monooxygenase lTTMTTMTTEQLGMNNSVNDKLqVLLIGAB l

Cyclohexanone

' Steroid

from

monooxygenase

monooxygenase

from

Pseudomonas

sp.

from Acinetobacter lSQKMDFnAIVIPGPFGPL

NCIMB 9872

calcoaceticus

CyIindrocalpon radicicola lAEWAEEFIJVLVVCJAPAGE

monooxygenase from Rhodococcus sp. lTA?TIH?VIJAVVIPACJFGEIYAVHK

NCIMB 9871

ATCC 11011

l

Cyclohexanone

ml

l

2-Oxo-4,5,5-trimethylcyclopent-3-enylacetyl-CoA monooxygenase lNRAKSPALIJAVVI($AEVTEIYQAFLINQAGMAVL

from

Pseudomonas

putida

NCIMB 10007

Type 2: FMN- and NADH-dependent l

3,6-Diketocamphane monooxygenase from lAMETGLIFHPYMRPGRSAAQTFDWGIK?A

Pseudomonas

putida

NCIMB 10007

l

2,5-Diketocamphane monoozqgenase IMQAGFFGTPYDIPTRTARQM

Pseudomonas

putida

NCIMB 10007

l

Luciferase

(1uxA) from Vibrio harveyi ATCC 33843 'M.K.F.G.N.F.L.L.T.Y.Q.P.P.E.L.S.Q.T.E.V.M.K.R.L.V.N.L.G

'

Luciferase

(1uxA) from Vibrio lMKFGNISFSYQPSGE

fischeri

ATCC 7744

l

Luciferase

(1uxB) from Vibrio IMKFGLFFLNFMFSKRSS

harveyi

ATCC 33843

l

Luciferase

(luxB) from Vibrio lM.K.F.G.L.F.F.L.N.F.Q.K.D.G.1

fischeri

ATCC 7744

from

‘indicates the N-terminal amino acid residue as determrned by sequence analysis.

TIBTECH FEBRUARY

1997 b'OL15)

58 f ecus

biotransformations have been used to yield chirdl synthons that have themselves been incorporated into successful chemoenzymatic syntheses of various potentially useful molecules. With the exception of the luciferases from I/i&o fischeri and Wtotobarterirlnl phophoreuml*, and the as yet uncharacterized activity in Cunt~ingharnella edlinlrlaral”, all other BVMOs examined, including pure enzyme preparations of various Type 1 and Type 2 enzymes, perform an enantiodivergent biotransformation of the racemic ketone bicyclo[3.2.0]hept-2-en-6-one to yield two homochiral lactones. This indicates that the two ketone enantiomers have been discriminated absolutely on a regio- plus enantioselective basis (Fig. 2a). Such a spectacular outcome has been confirmed using the separate substrate enantiomers with a pure preparation of CHMO (Ref. 21). Equally remarkable is that the outcome from 2,5-DKCMO represents a mirrorimage of the CHMO-catalysed biotransformation, thereby providing access to all four possible bicyclic [3.3.0] lactones in homochiral form (Fig. 2a). Interestingly, the outcome can be influenced significantly by the presence of chemical functionalities at either the 2 plus 3 or 7 plus 7’ positions’“, a valuable feature in developing active-site models of these enzymes (see below) These, and equivalent, biotransformations of various other unsubstituted bicyclic ketones containing a furan or pyran ring have been used to yield chiral synthons that can themselves be incorporated into chemoenzymatic syntheses of useful compounds as diverse as the pheromones multifidene and viridiene, the antibiotic sarkomycin A and the insect antifeedant clerodin (Fig. 2b). Currently, such biotransformations are undoubtedly the best way to achieve asymmetric Baeyer-Villiger oxidations. although recently some limited enantioselectivity has been achieved in equivalent chemical reactions catalysed by various expensive metal-based reagent+J7,

Kinetic resolutions including synthetic applications With other racemic substrates - including 2-bromo3-hydroxy-substituted [3.2.C)] ketones’“. various substituted [2.2.1] ketoneP and “-substituted monocyclic ketones”,” - both CHMO and the two DKCMO isozymes perform a very different type of regio- plus enantioselective biotransformation; each case yielding by kinetic resolution a single homochiral lactone (Fig. 3a). Changing the nature and/or length of the “-side-chain of the monocyclic substrates can have a significant effect on both the oucome and yield of these biotransformations”. Biotransformations of this type have been exploited to yield key chiral synthonr for chemoenzymatic syntheTes. Useful targets produced in this way have included potent antiviral carbocyclic nucleosides’“, a key precursor for the synthesis of the powerful insect antifeedant azadirachtin’s, and (g-5-hexadecanolide, the female sex pheromone of Kspa orientalir”‘. Another such application, the successful chemoenzymatic synthesis of the important coenzyme (R)TIBTECH FEBRUARY1997

(VOL151

lipoic acid, illustrates a number of points about the use of these enzyme? to perform useful biotransformations (Fig. 3b). Conventional retrosynthetic analysis identified a route to (R)-lipoic acid in which (a-methyl 6,8dihydroxyoctanoate, obtained indirectly from the appropriate regioplus enantioselective BVMOdependent biotransformation of racemic 2-(2’-acetoxyethyl)cyclohexanone, would achieve the desired objective’“. A BVMO from P putida, 2-0x0-4,5,5trimethylcyclopent-3-enylacetyl-CoA monooxygenase, performed a kinetic resolution with the required regioselectivity but the wrong enantioselectivity. Hydrolysis of the resultant (R)-(E)-lactone gave, the (R)-diol, which could be converted into the required additional chemical steps. (S)-diol by p e rf orming Further investigation3’ revealed that cyclopentanone monooxygenase from cyclopentanol-grown Pseudornoklas sp. NCIMB 9872, an enzyme previously noted for its poor enantioselectivity with various other ketone substrates, was able to yield the (S)-(E+lactone, thereby completing the original planned synthetic route.

Biotransformation

of mesuketones

Some highly selective biotransformations have also been recorded with prochiral ketone substrates such as l-methylcyclohexanone. This and other meso compounds, including various bi- and tricyclic ketones, were asymmetrized to homochiral lactone products (e.r. > 98%) by CHMO (Ref. 5). The ability of other BVMOs, including those from l? putida, to perform equivalent asymmetrizations has been confirmed by a recent study of various substituted cyclobutanones, which, interestingly, identified some substrates that were biotransformed by both CHMO and 2,5DKCMO with the same enantioselectivity”“. Some of these biotransformations have been exploited to yield chiral synthons with potential value for the chemoenzymatic synthesis of many useful compounds, including the antibiotic iononycin” and various lignan derivatives with interesting antileukaemic activities33.

Active site modelling of BVMOs To understand the exquisite stereoselectivity of BVMOs, and to make predictions of the likely products from untested substrates of commercial interest, it is necessary to know the three-dimensional structure of potentially useful biocatalysts such as CHMO and the DKCMO isozymes. To date, no such structures have been established. Full amino acid sequences are known only for CHMO from A. calroacetirus, although there are partial sequence data for various other enzymes (Box 1). Good progress is being made on a joint international programme to coordinate investigation into the three-dimensional structure of CHMO and the DKCMO isozymes; the parties involved are the universities of Exeter, UK (A. Willetts); London, UK (J. Ward); Edinburgh, UK (S. Chapman, G. Reid); Milan, Italy (G. Ottolina) and Brock, Canada (H. Holland). In the absence of any full structures, a

39

f ecus

a& +a:;;>0CHMO (Jy” 2,5-DKCMow -0 +(-:;;;. e.e.

>95%

>95%

e.e.

>89s

e.e.

299%

e.e.

/ b

I

111,,b

Viridiene

Multifidene

(4

?3-

0

““C02H

0

Sarkomycin

A

Clerodin Figure

2

Enantiodwrgent biotransformation of bicyclo[3.2.0]hept-2-en-6-one by cyclohexanone monooxygenase (CHMO)*” and 2,5-diketocamphane 1,2-monooxygenasezz. (b) Chemoenzymatic syntheses of various useful target molecules (multifidene and wndlene*3, sarkomycin A*4 and clerodin25) involving the blotransformatlon by (CHMO) of bicyclo[3.2.0]-type ketones. (a)

number of attempts have been made to develop models that explain the outcomes of biotransformations performed by these enzymes. Three very different approaches have been used but there is significant comparability between some outcomes in the case of CHMO from NCIMB 9872, the most widely modelled enzyme to date. ‘Cubic space’ models of BVMOs One approach has been to exploit some form of ‘cubic space’ approach3 to develop an active site model. It has been assumed that the outcome ofevents in the active site will be determined by a combination of the stereochemical and the stereoelectronic effects originally devised for the hypothetical enzyme mechanism”’ and the equivalent chemical reactior+. The model of CHMO proposed by Furstoss (Fig. la) has now been developed through several iterations to take account of the outcomes of an increasing number of ketone biotransformations’. It is based on a regular cubic space containing ‘forbidden zones’ that represent areas of cteric hindrance. The confines of the model are established by testing the theoretical positions that may be adopted by the transition state intermediates of any particular biotransformation, and matching these with the actual outcome.

A Gnilar approach has produced the Milan-Brock model of the active site of CHMO, which is probably the most realistic to date and the only one to explain the outcomeT of both sulfoxidatiorP3” and lactonization”‘biotransformations (Fig. -lb). In this case a molecular modelling program (Hyperchem) was used to minimize the total energy of each biotransformation product. The processed structures were then superimposed, either at the S atom along the S=O bond or the 0 atom along the C-O peroxide bond, to define various binding pockets that emerge from inserting the minimized structures inside a ‘cage’ initially composed of several cubes of 1.3-l k edge. The model can even explain the recorded outcomes with cyclic thiolactones. which are known to be suicide inhibitors of CHMO (Ref. 40). Mechanism-based models of BVMOs The other models that have been produced are more mechanisn-based. Taschner proposed such a model for CHMO by using a very different approach’. He argued that the active site of this flavoprotein is likely to be similar to that of human and Esdrcvicl~i~~ coli glutathione reductase. two other FAD-binding proteins for which the three-dimensional structure is already known. The N-terminal sequence ofall three proteins TIBTECH FEBRUARY 1997 (VOL 15)

60 f ecus

a 0 R (i

0

0

BVMO

.\oR e V-b),

W-U, (3

b OAc

I

BVMO

1

/

(q

\

BVMO2

~

OH OH (+)-(I?)-methyl 6,6-dihydroxyoctanoate

I

OR OR (+)-(a-

R = p- NO&H,CO

0

v

Meo-

OH OH

(S)-methyl 66dihydroxyoctanoate

s-s

I ~ I

~

(+)-(R)-lipoic acid Figure 3 monocyclic ketones by Baeyer-Villager monooxygenases (BVMO). (b) Chemoenzymatic synthesis of (RZ-(+)-lipoic acid by either 2-oxo-4,5,5-trimethylcyclopent-3-enylacetyf-CoA monooxygenase (BVMO 1; Ref. 29) or cyclopentanone monooxygenase (BVMO 2; Ref. 32).

(a) Kinetrc resolution of 2’substituted

is highly conservedand includesa similarBAB-domain incorporating the sameGxG,xxG FAD(=adenosine)binding motif’s, Taking this as a starting point, and then applying the same stereochemical and stereoelectronic considerationsusedto formulate the ‘cubic space’models, Taschner developed a model that successfully explained the observed outcomes from various lnesoketones (Fig. 3~). A key feature is that the hydroperoxide of the oxygenated FAD intermediate was assumedto be attached to the re-face of the isoalloxane ring ofthe flavin cofactor, and that the ketone TlBTECHFEBRUARY1997lVOL151

substrate approaches the hydroperoxide from the direction of the dimethylbenzene moiety. This mechanism-based model is compatible with those developed by the alternative ‘cubic space’approach. Kelly has proposed a second mechanism-based model (Fig. 4d), developed initially from the outcome of the biotransformation of a single, unsubstituted tricyclic ketone by CHMO (Ref. 41). A basicdifference between this model and the others is that it is proposed that enantioselectivity is controlled exclusively by stereoelectronic considerations,being a function of the diastereofacialselectivity of the enzyme for the substrate. The likely influence of stereochemical considerations imposed by the interaction of the ketone substrate and the confines of the active site was not considered, despite the existence of data that clearly demonstrate the dramatically different outcomes of the biotransformation of unsubstituted versus substituted bicyclic [3.2.0] ketones (vide S~JVU).In another substantial departure from Taschner’smodel, it was assumed that, in the transition state intermediate formed in the active site of CHMO, the hydroperoxide is attached to the xi-face of the isoalloxane ring of FAD, and that the ketone substrate approaches the hydroperoxide from the direction of the diazine moiety. The resultant transition state intermediate was described as having an ‘S-migration configuration’ when the Cahn-Ingold-Prelog rules were applied. It was proposed that the enantioselectivity of the resultant biotransformation was dictated by this ‘S-migration configuration’. When extended to the outcomes of equivalent biotransformations, both DKCMO isozymes were defined as establishing ‘R-migration configuration’ transition stateintermediates, thereby yielding a different lactone product. Kelly’s model has its merits asan explanation for observed outcomes from the tricyclic ketone substrate, but fails to explain both outcomes of sulfoxidation by these enzymesl* and why CHMO and the DKCMO isozymesbiotransform somesubstitutedcyclobutanones with identical enantioselectivityss. Concluding remarks A comprehensive explanation of some of the bizarre selectivities observed with this interesting group of enzymes must await the characterization of the relevant three-dimensional structures. However, the values of these enzymes in asymmetric synthesis, enabling convenient accessto chiral lactones and sulfoxides, has been clearly demonstrated. Further applications of this emerging field of biotransformations will undoubtedly follow. Although no largescale syntheseshave been commissioned to date, a number of the enabling technologies, including the relevant coenzyme recycling methodologies, have been successfully developed already: further issues, including the important matter of ensuring adequate oxygen availability to optimize the biotransformations, are currently being addressedby an on-going research programme involving the UK universities of Exeter and Liverpool, and University College London.

61

f OCUS

Preferential formation of (1 S,5R)-lactone

Non-permitted formation of (1 SW?)-lactone

b

Top

Preferential formation of (1 R,5S)-lactone

Non-permitted formation of (1 R,5S)-lactone

Active site model of CHMO from lactonization data

Side

H CR)

Flavin

Configurational

HO

correlation to explain the outcome of the biotransformation bicyclo[3.2.0]hept-2-en-6-one by CHMO

of

d R’O R’OOH

Non-migrating R2,0”,. R”

o-o

b

Migrating

group

Migrating

R’,

group

@”

k

group

Non-migrating

group

? R’/”

R’ = flavin coenzyme R2 = H

2,5-DKCMO 3,6-DKCMO (R)-migration configuration

CHMO (S)-migration configuration

Figure 4 Active site models of cyclohexanone monooxygenase (CHMO). (a)‘Cubic space’ model proposed by Furstoss et a/.1: ‘the forbidden zone’ is outlined by the dotted lines. (b) ‘Cubic space’ model proposed by Ottolina et al. 37-39(c) Mechanism-based model proposed by Taschner et al.5 (d) Mechanism-based model of Kelly”1842.

References 1 Alphand, V. and Funtoss, R. (lYY5) m Eqwe C‘~r~ilyv< !,I Ogyuri .Synfhws (Urauz, K and Waldmann, H.. eds). pp 7-15-772. VCH 2 Fried, J Thorna, K. W. and Khngsbrrg, ‘4. (lYj3)J .%n. Chern. Sot 75. 5764-5767 3 Baeyer, A. and Vllhger, V. (1899) &I. Dtvh. Ciro,~. 32. 3625-3633 4 Latham, J. A. and Walsh. C. (1987)]. .+,I. Ciwn. Sor 1119,3421-3-127 5 Taschner, M. J., Peddada. L.. Cyr, P.. Chen, Q-Z. and Black, D. J. (1992) in .Mrmbiai Reaymrr iti Ogar~rr .Syrltiw.G (Serve. S., cd.), pp. 347-360, Kluwer Acadennc

10 Secundo, F . Carrra. G.. Rwa, S.. Batnstrl. E. and B~anclx. D. (1993) B~o/ctl~rd ht. 13, 865-8711 11 Pasta, P.. Carrea, G Gaggero, N.. Grogan. G. and Wllletts, .4. (1996) Biotctkrioi ht. 1 H. I 123-l 1% 12 Wlllrtts. A.. Knowle<, C J.. Lrwtt. M. S., Roberts. S. M., Sandey. H. and Shlpston. N. F. (199lj.I C/rent Soi.. Prrkirz Trans. 1 1608~1611l 13 Donoghur, N. A.. Noms, D. B. and Trudgill, P. W. (lY76) Eur.]. B~dm 63, 175-192 14 Chen. Y-C. J., Proplcs. 0 P. and Walsh, C. 7. (1988)]. Bacfcnol. 170, 781&7XY 15 Wwenga, K. K., l)rwth. J. and Schulz, G. E. (1983) .\ .\fo1 Biol. 167, 725-73’1

17 Zqler.

M. M. and Haldwn,

T 0. (1981) Cw. To11,Bioq

12.65-l

13

TIETECH FEBRUARY 1997 lVOL151

Designing and optimizing library selection strategies for generating high-affinity antibodies Hennie R. Hoogenboom Since its invention at the beginning of the 199Os, antibody phage display has revolutionized the generation of monoclonal antibodies and their engineering. It is now possible to create antibodies binding to any chosen target antigen without the use of laboratory animals or hybridomas, In a system that completely by-passes the immune system. Making antibodies

from single-pot phage libraries, and

improving their affinity up to the picomolar range if necessary, has never appeared easier. In this review, a variety of phage library-based strategies for the isolation of high-affinity antibodies are presented.

The biotechnological generation of high-affinity monoclonal antibodies has traditionally involved the production of hybridomas from spleen cells of immunized animals. Now the use of phage antibodies offers a new route for the generation of antibodies, including antibodies of human orrgm, which cannot be

TIBTECH FEBRUARY1997(VOL151

Copyright0

1997,

Elsewr

Suence

easily isolated by conventional hybridoma technology. Over the last decade, protein engineering methods to generate more human-like antibodies have been developed’ (chimerization and humanization of rodent antibodies) and, more recently, transgenic mice expressing human antibodies were proposed (reviewed in Ref. 2). ln all cases, antibodies are created or engineered with their specificity and affinity shaped by the mmlune system. With phage display, antibodies can be made completely in uirvo, by-passing the immune system and the immunization procedure, and allowing irl Ltd All rights reserved.

0167

- 7799/97/$17

00. PII: SO167-7799(96)10070-6