This document discusses the use of enzymes in the synthesis of bioactive compounds. It begins by noting the advantages of enzymes as catalysts such as their selectivity and ability to work under mild conditions. However, their narrow substrate specificity and instability have limited their application. Advances in biocatalysis over the last 20 years have helped address these drawbacks through immobilization, use of enzymes in non-aqueous solvents, and directed evolution. The document then discusses various applications of enzymes including in non-aqueous solvents, immobilization, directed evolution to modify substrate specificity and stability, synthesis of carbohydrates and chiral drugs, and their use in combinatorial chemistry.

1. Enzymes in the synthesis of bioactive compounds
Introduction
 Enzymes catalyze reactions with remarkable rate accelerations
over background
 They are chemoselective and regio- and stereospecific;
however, they usually present narrow substrate specificity limiting their
application.
 Enzymes are environmental friendly catalysts since they work in aqueous
solutions and at moderate temperatures, but their poor stability under in
vitro reaction conditions can become a strong limitation to their use in
chemical synthesis.

2. Besides the unique properties of enzymes as catalysts, synthetic chemists
have been reluctant to employ them for a number of reasons:
(i)most organic compounds are water-insoluble,and the water removal is
tedious and expensive;
(ii)limited availability of biocatalysts with the desired activity and substrate
specificity;
(iii) poor stability of the enzymes, and
(iv) relatively high cost of the biocatalyst
These drawbacks have been, at least partially, solved by the advances
produced in the biocatalysis field over the last 20 years.
The use of immobilized enzymes present two main benefits (i) easy separation of
enzyme from the reaction mixture; and (ii) the biocatalyst can be reused several times.

3. 2. General aspects of biocatalysis
1. Enzymes in non-aqueous solvents
For a long time enzymes were believed to work efficiently only in aqueous
solutions. Consequently, their utilization in organic synthesis was rather scarce
since the low water solubility of many substrates represented a serious obstacle.
The methods adopted evolved from the mixtures of water and water-miscible
organic solvents, biphasic aqueous-organic systems, reverse micellar systems,
and finally in nearly anhydrous organic systems.
Because the latter is attractive, it has undergone a rapid expansion among the
synthetic chemists during the last two decades.
Ionic liquids are a new class of non-aqueous solvents with non-molecular, ionic
character. These solvents are salts that are liquid at room temperature. Through
the choice of cations and anions, the physical and chemical properties can be
optimized for each application. The replacement of organic solvents by ionic
liquids can lead to remarkable improvements regarding reactivity, selectivity and
stability of the enzyme.

4.  An immobilized enzyme is an enzyme attached to an inert, insoluble material—
such as calcium alginate (produced by reacting a mixture of sodium
alginate solution and enzyme solution with calcium chloride). This can provide
increased resistance to changes in conditions such as pH or temperature.
 It also lets enzymes be held in place throughout the reaction, following which
they are easily separated from the products and may be used again - a far more
efficient process and so is widely used in industry for enzyme catalysed reactions.
2.2. Enzyme immobilization
Structure of Calcium alginate

5. Alginic acid, also called algin, is a naturally occurring,
edible polysaccharide found in brown algae. It is hydrophilic and forms a
viscous gum when hydrated. With metals such as sodium and calcium, its
salts are known as alginates. Its colour ranges from white to yellowish-
brown.

6. 3. From enzyme over-expression to tailor-made biocatalysts
1. The PCR revolution and the biocatalysis
 This has made possible to carry out enzymatic reactions that otherwise are
difficult to perform by chemical methods, like the synthesis or modification of
complex molecules such as carbohydrates, asymmetric carbon–carbon bond
formation, kinetic resolution of racemic epoxides or dioxigenation of aromatic
compounds.
 Microorganisms living under extreme conditions of temperature, pH, salt
concentration or pressure that are difficult or even impossible to be cultured,
provide a large number of new enzymes. The new DNA techniques make possible
to express enzymes from these organisms in others that can be cultured under
controlled conditions such as Escherichia coli.
 Therefore, extremophilic microorganisms are providing biocatalysts able to work
under conditions similar to those needed in organic syntheses.

8. In vitro enzyme evolution has been successfully used to modify enzyme features that
are of interest in organic synthesis, such as substrate specificity, optimum pH,
stereospecificity or enantioselectivity; this approach has also been applied to improve
enzyme stability at high temperature or in organic solvents.
2.3.2. Catalytic antibodies: tailor-made biocatalysts
Jencks suggested at the end of the 1960s, that stable molecules resembling
the transition state of a reaction might be used as haptens to elicit antibodies with
tailored catalytic activities and selectivities. Since the first reports by the groups of
Lerner and Schultz in the middle 1980s, more than 100 reactions have now been
successfully accelerated using catalytic antibodies (abzymes), including pericyclic
processes, group transfer reactions, additions and eliminations, oxidations and
reductions, aldol condensations, and miscellaneous cofactor-dependent
transformations.

9. However, the catalytic efficiency of most catalytic antibodies generated to date is
lower than that of their enzymatic counterparts. The substrate specificity of these
antibodies is broader than that observed with any natural aldolase and they are able
to catalyze a wide variety of intermolecular aldol reactions between ketone–ketone,
ketone–aldehyde aldehyde–ketone and aldehyde–aldehyde, as well as several
intramolecular aldol condensations.

10. 3. Enzymes in the synthesis of chiral drugs
Chirality plays a crucial role in nature and it is a key factor in the efficacy of many drugs.
For achieving these transformations different classes of enzymes have
been exploited.
The most often used ones are:
(i)hydrolases (lipases, esterases proteases/amidases), for the synthesis of esters, acid,
peptides/amides;
(ii) dehydrogenases, for the oxidation of alcohols and reduction of carbonyls;
(iii) mono- and dioxygenases for hydroxylation of unactivated carbon;
iv) aldolases for the stereoselective condensation of carbonyl compounds; and
(v) oxynitrile lyase for synthesis of chiral hydrox nitriles

11. However, when only one of the enantiomers is required, the enzymatic resolution of
racemic substrates has the limitation that the maximum yield is 50%.
There are several ways to overcome this problem:
(i) the use of meso compounds or prochiral substrates;
(ii) stereoconversion of the remaining enantiomer; and
(iii) dynamic kinetic resolution.
Enantiomeric excess (ee)

12.  They showed that the isomerization of 1-phenylethyl alcohol (Fig. 4) by a
ruthenium catalyst and enzymatic acylation with 4-chlorophenyl acetate as acyl
donor resulted in transformation of the racemic alcohol to enantiomerically pure
acetate.
 In this example the ruthenium catalyst promotes the racemization of
the alcohol substrate. Racemization involves abstraction of the alpha-proton to
give a ketone intermediate and subsequent readdition of hydrogens to the ketone.

13.  Mischitz and Faber studied the asymmetric
nucleophilic opening of ()-3-methyl-2-
pentyloxirane (rac-1, Fig. 5) catalyzed by a
crude immobilized enzyme preparation from
Rhodococcus sp. (NOVO SP 409).
 The reaction in aqueous buffer gave the diol
(S)-2 in only 40% ee.
 However, when the enzymatic hydrolysis of ()-1
was carried out in the presence of the non-
natural nucleophile azide, the (S)-diol, 2, and
(R)-azidoalcohol, 3, were obtained in >90%
and 60% ee respectively (Fig. 5).
 Therefore, a simultaneous and opposite
enantio-discrimination of two nucleophiles
(water and azide) was observed.
 This parallel kinetic resolution afforded the (S)-
diol product in higher ee.

14. 4. Enzymatic synthesis of carbohydrates: new tools for new drugs
Carbohydrates have multiple hydroxyl functions of similar chemical reactivity;
for example, five monosaccharides can be linked together to form linear or
branched chains that can give rise to 32million different compounds. Therefore,
a special knowledge of selective reactions and protecting groups are needed
to achieve successfully their synthesis and modification.
1. Aldolase enzymes for C–C bond formation
Aldolases have attracted the interest of organic chemists because their ability to
catalyze the formation of C–C bonds by an aldol addition reaction between an
aldehyde and a ketone, with a high degree of stereochemical control.

15.  Dihydroxyacetone phosphate (DHAP) depend
ent aldolases produce 2-keto-3,4-dihydroxy a
dducts and, with some exceptions, they cont
rol the configuration of the newly formed ste
reogenic centers.
 An additional advantage of these enzymes is
that they are stereocomplementary, that is, t
heir use allows the synthesis of the four poss
ible diaestereoisomers for a given pair of sub
strates (Fig. 6).
 DHAP-dependent aldolases have shown their
utility in the synthesis of
carbohydrate, carbohydrate-like structures or
non-carbohydrate compounds.

16. Aldolases provided adducts that are not only densely but also differentially
functionalized. Thus, four different state of carbon oxidation can be found in four
contiguous carbons.
This substitution pattern allows transformation of aldol adducts to a variety of
complex natural products (Fig. 7).

17. 4.2. From glycosidases to glycosynthases
Glycosidases are readily available enzymes and use simple glycosyl donors, that
can be even the free monosacharide.
These hydrolytic enzymes are able to catalyze the formation of glycosidic bonds
in a stereospecific manner.
However, when applied to the synthesis of di and oligosaccharides the problem
of the regioselectivity has to be faced. In contrast to lipase and protease that work
in anhydrous organic solvents, glycosidases do not.

18. Several ways have been applied to improve yield and to control the regioselectivity of glycosid
ase-catalyzed reactions
(ii) Use of very reactive glycosyl donors, in such a way that it is cleaved more rapidly than the
product formed. Under these conditions, the reaction is kinetically controlled.
(ii)Introduction of substituents at certain positions in the sugar acceptor. For instance, it has
been shown that both the nature of the substituent and the anomeric configuration influence
the regioselectivity and the yield of the glycosidation.
(iii)Manipulation of the medium, using organic cosolvents to reduce the total volume of water.
Also, addition of salts at high concentration has been used in order to reduce the water activity.
The use of lipid-coated glycosidases improves the behavior of the enzyme in media with low w
ater activity.

19. 5. Enzymes enter the field of combinatorial chemistry
 Arecent approach in combinatorial chemistry is to create smaller and focuses libraries.
 Thus, combinatorial chemistry is used to generate a high degree of structural and chemical
diversity on initial lead compounds in order to optimized them for
binding, target specificity, bioavailability, etc.
 Biocatalysts can be employed in two different combinatorial schemes to gener
ate libraries of compounds:
 (i) combinatorial biocatalysis, which assembles in vitro new compounds and
derivatives using isolated enzymes and
(ii) combinatorial biosynthesis, which shuffled or modified the genes of natural
biosynthetic pathways to produce in vivo libraries of ‘unnatural’ natural products.

20. 5.1. Enzymes in polymer-supported synthesis
 Combinatorial chemistry often involves the synthesis on polymeric supports
.
 In polymer-supported synthesis the substrate is anchored to the polymer th
rough a functional group, developed, involving the use of hydrolases such
as protease, lipase or acylase.
 Waldmann et al.
described the exo-linker 1, which comprises a 4-acyloxy-3-carboxy-benzylox
y group (Fig. 10). The linker 1 is attached as an amide to the solid phase. It
contains an acyl group, for example acetate, which can be cleaved
by lipases or estereases. Cleavage of the acyl group by a lipase generated a
phenolate 2, which fragments to give a quinone methide 3 and releases th
e desired product 4.
 The quinone remains attached to the solid phase and is
trapped by water or an additional nucleophile.

21. 5. Enzymes enter the field of combinatorial chemistry
 Arecent approach in combinatorial chemistry is to create smaller and focuses libraries.
 Thus, combinatorial chemistry is used to generate a high degree of structural and chemical
diversity on initial lead compounds in order to optimized them for
binding, target specificity, bioavailability, etc.
 Biocatalysts can be employed in two different combinatorial schemes to gener
ate libraries of compounds:
 (i) combinatorial biocatalysis, which assembles in vitro new compounds and
derivatives using isolated enzymes and
(ii) combinatorial biosynthesis, which shuffled or modified the genes of natural
biosynthetic pathways to produce in vivo libraries of ‘unnatural’ natural products.

22. 5.1. Enzymes in polymer-supported synthesis
 Combinatorial chemistry often involves the synthesis on polymeric supports
.
 In polymer-supported synthesis the substrate is anchored to the polymer th
rough a functional group, developed, involving the use of hydrolases such
as protease, lipase or acylase.
 Waldmann et al.
described the exo-linker 1, which comprises a 4-acyloxy-3-carboxy-benzylox
y group (Fig. 10). The linker 1 is attached as an amide to the solid phase. It
contains an acyl group, for example acetate, which can be cleaved
by lipases or estereases. Cleavage of the acyl group by a lipase generated a
phenolate 2, which fragments to give a quinone methide 3 and releases th
e desired product 4.
 The quinone remains attached to the solid phase and is
trapped by water or an additional nucleophile.

23. Figure 10. Principle for the development of the
enzyme-labile 4-acyloxybenzyloxy linker group
Figure 11. General strategy for the liquid-phase synthesi
s of disaccharides using glycosidases

25. 5.2. Combinatorial biocatalysis
The creation of focused libraries is based on derivatization of existing molecules.
This approach mimics the chemistry that occurs in biological systems where
precursors are modified by the action of biocatalysts.
For modification of existing lead compounds, the special properties of enzyme as
catalysts report some advantages over synthetic chemical reactions:
(i)the high chemo-selectivity of enzymatic reactions allows modifying only one kind
of functional groups in a scaffold molecule that typically contains several different
functional groups.
(ii)the enzyme regioselectivity provide the opportunity for specific combinatorial
modification of lead molecules with multiple copies of the same functional group, for
example, glycosylation catalyzed by glycosyltransferases with different
regioselectivity.
(iii)the enzyme stereocontrol over the reaction permit a combinatorial approach to
the three-dimensional structure of the compound, for example, using the four DHAP-
dependent aldolases.

26. These biocatalytic reactions are available for
can be grouped in three mayor categories
(i) introduction of new functional groups
(ii) modification of existing functionalities
(iii) addition onto functional group
An important issue regarding combinatorial bicatalysis is that of orthogonality.
Modification of a substrate by one enzyme, ‘A’, may prevent it from being
substrate for another enzyme, ‘B’, while the modification of the initial substrate by
‘B’, may not preclude it from being a substrate for the first enzyme, ‘A’ (Fig. 12)
Figure 12. Orthogonality of biocatalysts

27. A first generation of derivatives is
modified by another round of
biocatalytic reactions at additional
reactive sites to produce a second
generation of derivatives. After several
iterations is possible to create a great
number of derivatives from the original
lead compound (Fig. 13)
Figure 13. Iterative synthesis of a 600-member library from
the flavonoid bergenin

28. Figure 14. Production of sugar-
nucleotides and oligosaccharides by
combined use of C. ammoniagenes with
E. coli strains metabolically engineered.
(A) Production system for UDP-Gal and
globotriose.105 (B) Production system for
CMP-NeuAc and 30-sialyllactose.

29. Figure 15. The erythromycin PKS system. The pathway has three PKS polypeptides containing six
modules and loading and releasing domains. The cyclized structure shown needs to be further modified
to bring erythromycin to its final structure.

30. Figure 16. Two different strategies for the synthesis of ‘unnatural’ natural products by combinatorial biosynthesis.
(A) Precursor-directed polyketide biosynthesis; inactivation of the first ketosynthase (KS) prevent the production of
the second-step precursor, allowing to prime the second module with synthetic analogues of the product of the
first module.113a
(B) Introduction of auxiliary catalytic domains; by replacing the ketoreductase (KR) domain of module 2with a KR-
DH(dehydratase)-ER(enoylreductase) tridomain from the rapamycin synthase ( ) new chemistry is introduced
into the reaction sequence catalysed by the module

31. Chemical Biology

32. Introduction
Chemical biology and its historical roots
 According to Nature, chemical biology as both the use of chemistry
to advance a molecular understanding of biology and the harnessing
of biology to advance chemistry

33. The history of nitrous oxide
Joseph Priestley (1733–1804) began to perform his own experiments on gases, referred to
as „airs‟in the parlance of the time2. Priestley, perhaps most identified with the discovery of
oxygen, also isolated at least ten other gases, including nitrous oxide. Using biology to
advance chemistry, Priestley incubated mice with these gases.
The approach provided a primitive characterization method (upon exposure to a compound,
does a mouse live or die?) that has since fallen into disfavor with synthetic chemists.
Turning the tables on Priestley‟s approach to chemical biology by using chemistry to advance
biology, Sir Humphrey Davy‟s experi-ments with newly isolated, unfamiliar gases omitted the
mouse entirely. In what must be considered an act of either lunacy or egotism, Davy (1778–
1829) carried out his experiments on himself. Not surprisingly, his experiments with carbon
monoxide almost proved fatal. In one experiment, Davy inhaled four quarts of nitrous oxide
isolated in a silk bag. The pleasant intoxicating effect of the gas inspired Davy to name it
laughing gas.

34. Davy had noted the possible advantages of using nitrous oxide in surgical procedures,
medical uses for the gas remained unexplored until experiments by the American dentist
Horace Wells in 1844 (ref. 5).Application of the chemistry of nitro-gen oxides to advance
biology and medicine.
During an attempt to synthesize ammo-nium cyanate, Wöhler (1800–1882) heated a
solution of silver cyanate and ammonium chloride. Separately, he also heated lead cya-
nate and aqueous ammonia. In both cases, he obtained not the expected product, but urea
(Scheme 1). Wöhler‟s synthesis showed that inorganic starting materials could be used to
synthesize substances previously associated only with living organisms. In other words,
chemical synthesis requires no „living‟or „vital force‟ to make biologically active
compounds.

35. Scheme 1 Wöhler’s synthesis of urea. Yielding a chemical previously isolated only
from man or dog, the synthesis of urea represented a landmark achievement in
organic synthesis and chemical biology.
Chemical synthesis requires no „living‟or „vital force‟ to make
biologically active compounds.

36. About 91-96% of urine consists of water. The remainder can be broadly
characterized into inorganic salts, urea, organic compounds, and
organic ammonium salts
The total solids in urine are on average 59 g per person per day. Organic
matter makes up between 65% and 85% of urine dry solids, with volatile
solids comprising 75–85% of total solids. Urea is the largest constituent of
the solids, constituting more than 50% of the total.

37. Ehrlich experimented with the newly discovered aniline dyes derived from
coal tar and found that some dyes could differentially stain specific cells and
tissues. He correctly surmised that this difference resulted from a chemical
reaction of the dyes with specific substances within the cells. For example,
the predominant basicity of dyes capable of staining cell nuclei led Ehrlich
to term the nucleus “basophilic.” Applying the aniline methylene blue dyes
for diagnostics, Ehrlich identified a tiny rod-shaped bacterium as the culprit
responsible for tuberculosis.

38. Privileged Structures and Drug-like Molecules
Certain scaffolds/supports are capable of binding to multiple
receptor targets
Appropriate structural modifications can change the activity
Privileged structure
benzodiazepines
(a) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.; Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P.
S.; Chang, R. S. L.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J. J. Med. Chem. 1988,
31, 2235. (b) Ariëns, E. J.; Beld, A. J.; Rodrigues de Miranda, J. F.; Simonis, A. M. in The Receptors: A Comprehensive Treatise;
O'Brien, R. D., Ed.; Plenum Press: New York, 1979, p. 33. (b) Ariëns, E. J. Med. Res. Rev. 1987, 7, 367. (c) Patchett, A. A.; Nargund, R.
P. Annu. Rep. Med. Chem. 2000, 35, 289. (d) Hajduk, P. J.; Bures, M.; Praestgaard, J.; Fesik, S. W. J. Med. Chem. 2000, 43, 3443. (e)
Fecik, R. A.; Frank, K. E.; Gentry, E. J.; Menon, S. R.; Mitscher, L. A.; Telikepalli, Med. Res. Rev. 1998, 18, 149. (f) Horton, D. A.;
Bourne, G. T.; Smythe, M. L. J. Computer-Aided Mol. Des. 2002, 16, 415. (g) Klabunde, T.; Hessler, B. Chembiochem. 2002, 3, 928. (h)
Matter, H. Baringhaus, K.H.; Naumann, T.; Klabunde, T.; Pirard, B. Comb. Chem. High Throughput Screen. 2001, 4, 453. (i) Horton, D.
A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893.

39. • Indicated for treatment of
anxiety, seizure, insomnia,
panic disorder, and alcohol
withdrawal.
Lorazepam (Ativan)
• The original date-rape drug,
and the origin of the term
“roofie”
• One of only two drugs in the
U.S. for which a first
possession charge is a
mandatory felony … the other
is crack cocaine.
Flunitrazepam (Rohypnol)

40. Drug-likeness
Only 32 scaffolds describe half of all known drugs.1
Average number of side chains per molecule is 4.2
Drug-likeness may be an inherent property of some
molecules.3
Try these first.
1 Bemis, G. W.; Murcko, M. A. J. Med. Chem. 1996, 39, 2887.
2 Bemis, G. W.; Murcko, M. A. J. Med. Chem. 1999, 42, 5095.
3 Ajay; Walters, W. P.; Murcko, M. A. J. Med. Chem. 1998, 41, 3314.

41. Many nondrug-like molecules show up as active in
screens - false positives.
Appear to bind to numerous receptors, but not at
the site of the natural ligand (called promiscuous
drugs)

42. Structural Modifications
Increase potency
Increase therapeutic index - measure of the ratio of the
concentration of a drug that gives undesirable effects to
that which gives desirable effects
e.g., LD50 (lethal/deadly dose for 50% of the test animals)
(effective dose to give maximum effect in
50% of test animals)
ED50
Therefore, want LD50 to be large and ED50 to be small.
The larger the therapeutic index, the greater the margin
of safety.
The more life threatening the disease, the more
acceptable is a low therapeutic index.

43. Types of Structural Modifications
Homologation - changing compound by constant unit (e.g., CH2)
Effect of carbon chain
length on drug potency
Pharmacokinetic explanation: Increasing chain length increases
lipophilicity and ability to cross membranes; if lipophilicity is too
high, it remains in the membrane
Pharmacodynamic explanation: Hydrophobic pocket increases
binding with increasing length; too large and does not fit into
hydrophobic pocket
Figure 2.18

44. Table 2.8. Effect of Chain Length on Potency. Antibacterial activity of 4-n-alkylresorcinols
and spasmolytic activity of mandelate esters.
OH
R
OH
OH
CHCO2R
R Phenol coefficient % Spasmolytic activitya
methyl — 0.3
ethyl — 0.7
n-propyl 5 2.4
n-butyl 22 9.8
n-pentyl
in throat
33 28
n-hexyl 51 35
n-heptyl 30 51
n-octyl 0 130
n-nonyl 0 190
n-decyl 0 37
n-undecyl 0 22
i-propyl — 0.9
i-butyl 15.2 8.3
i-amyl 23.8 28
i-hexyl 27 —
lozenges
Branched chain
groups are less
lipophilic than
straight chain
groups. 7

45. Ring-Chain Transformations
Transformation of alkyl substituents into cyclic
analogs often does not affect potency.
Chlorpromazine (antipsychotic)
(2.40, X = Cl, R = CH2CH2CH2NMe2) and
(2.40, X = Cl, R = CH2CH2CH2N )
have equivalent tranquilizing effects
Ring-chain transformation can have pharmacokinetic
effects, such as increased lipophilicity or decreased
metabolism.

46. Bioisosterism
Bioisosteres - substituents or groups with
chemical or physical similarities that produce
similar biological properties. Can…
…attenuate toxicity
…modify activity of lead
…and/or alter pharmacokinetics of lead.

47. Table 2.2
1. Univalent atoms and groups
3 2
a. CH NH OH F Cl
b. Cl PH2 SH
c. Br i-Pr
d. I t-Bu
2. Bivalent atoms and groups
a.
b.
NH
CONHR
CH2
COCH2R
O
CO2R
S
COSR
Se
3. Trivalent atoms and groups
a.
b.
CH N
P As
4. Tetravalent atoms
C
a. Si
b.
5. Ring equivalents
a.
b.
c.
CH CH
CH
O
S (e.g., benzene, thiophene)
(e.g., benzene, pyridine)
CH2
N
S NH (e.g., tetrahydrofuran,
tetrahydrothiophene,
cyclopentane, pyrrolidine)
C N P
Classical Isosteres
Table 2.9

48. Table 2.10
Do not have the same
number of atoms and do
not fit steric and
electronic rules of
classical isosteres, but
can have similar
biological activity.
Non-Classical Isosteres

50. 10. Azomethine
11. Pyridine
12. Benzene
13. Ring equivalents
14. Spacer group
N
CN
C
N
NO2
N+
R NR3
+
N N
N S
N
S O
N
O
N
O
N
H
N
R N
O R'
N
O R'
R
R
R
H3C
O
H
N O
R
O
N
N
H
R
H
NH2
(CH2)3
15. Hydrogen
H F

51. Example of
Bioisosteric
Analogues
Table 2.11
N
O
CH3
X
CH3O
O
N N
N
3
O
CH
Cl
OH
Y
Z
N
H
X = OH (indomethacin)
= NHOH
=
3
Y = CH O Z = Cl
Z = SCH3 (sulindac)
Y = F
Anti-inflammatory agents

52. Changes in Activity by Bioisosterism
If the S in phenothiazine neuroleptic drugs (2.40) is
is replaced by -CH=CH-/-CH2-CH2- bioisosteres,
then dibenzazepine antidepressant drugs (2.43)
result.

53. Changes resulting from bioisosteric replacements:
Size, shape, electronic distribution, lipid solubility, water
solubility, pKa, chemical reactivity, hydrogen bonding
Effects of bioisosteric replacement:
1. Structural (size, shape, H-bonding are important)
2. Receptor interactions (all are important)
3.Pharmacokinetics (lipophilicity, hydrophilicity, pKa,
H-bonding are important)
4. Metabolism (chemical reactivity is important)
Bioisosteric replacements allow you to tinker with whichever
parameters are necessary to increase potency or reduce toxicity.

54. Bioisosterism allows modification of
physicochemical parameters
Multiple alterations may be necessary:
If a bioisosteric modification for receptor
binding decreases lipophilicity, you may have
to modify a different part of the molecule with
a lipophilic group.
Where on the molecule do you go to make the
modification?
The auxophoric groups that do not interfere with binding.

55. Combinatorial Chemistry
Synthesis or biosynthesis of chemical libraries of
molecules for lead discovery or lead modification
Libraries prepared in a systematic and repetitive
way by covalent assembly of building blocks to
give diversity within a common scaffold.
Synthesis carried out on a solid support (polymer
resin).
Isolation and purification of product of each step
done by filtration and washing. Everything not
attached to polymer is washed away, which allows
use of excess reagents.

56. Alternatives: Carry out reactions in solution with
excess reagents, which are scavenged with a
polymer-bound scavenger. Filtration removes
excess reagents bound to polymer.
Or, use polymer-bound reagents.
Main differences among various methods of
combinatorial library synthesis:
1. Solid support used
2. Methods for assembling the building blocks
3.The state (immobilized or in solution) in which
libraries are screened
4.Manner in which structures of active compounds
are determined (encoding methods)

57. N = bx
# synthetic steps
# compounds
in library
# building blocks
in each step
If the # of building blocks varies in each step, then
N = bcd...
For example, consider a 4-step synthesis. If 25
different reactants (building blocks) are used in the
first two steps, 20 in step three, and 15 in step four,
there would be 25  25  20  15 = 187,500 different
compounds (from only 85 different building blocks).

58. Combinatorial Chemistry

59. Peptide Libraries – by Split Synthesis
Also called mix and split or split and pool method
Most common lead discovery approach for large
libraries (104 - 106 compounds), assayed as library
mixtures.
Produces a collection of polymer beads, each containing
one library member (100-500 pmol)
A combinatorial library of all pentapeptides comprised
of the 20 common amino acids: 205 = 3.2  106 peptides.

60. Identification of the Active Part
of the Lead
First consider pharmacodynamics
Pharmacophore - the relevant
groups on the compound that
interact with the receptor and
produce activity
Auxophore - the rest of the molecule

61. Some of the auxophoric groups interfere with
binding of pharmacophoric groups and must be
excised/removed.
Some of the auxophoric groups may neither bind
to the receptor nor prevent the pharmacophoric
groups from binding - these can be modified
without effect on potency.
Modification of these auxophoric groups may be
used to correct
absorption, distribution, metabolism,
and excretion", and describes the disposition
of a pharmaceutical compound within

62. One approach to determine
…which are pharmacophoric atoms
…which are auxophoric atoms hindering binding
…which are auxophoric atoms that can be modified
is to cut away pieces of the lead and measure the
effect on potency.

63. Example of Pharmacophore and
Auxophore Identification
Assume the well-known addictive analgesics below are
the lead compounds. The darkened part is known to be
the pharmacophore (you would not know that in a real-
life example).
All bind to the Mu () opioid receptor
What happens if we excise the bridging O (shown above
not in the pharmacophore)?

64. 3-4 times more potent than morphine
Note: structure drawn to mimic that of
morphine; it is not the lowest energy
conformer.
Maybe additional conformational flexibility
allows the molecule to bind more effectively.

65. Next, remove part of the cyclohexene ring (also
not in the pharmacophore).
Less potent than morphine, but much
lower addictive properties.

66. Next, cut away the methylene of the other cyclohexane ring.
about the same potency
as morphine
Demerol
10-12% the potency of
morphine…
…but analog synthesis
is much easier

67. If the piperidine ring is cleaved, the acyclic molecule
dextropropoxyphene (2.26, Darvon) has half to two-thirds
the potency of codeine.
Note: Binds to Mu opioid receptor,
like morphine.
Molecule is still drawn in the shape of
morphine; otherwise would not know
it mimics morphine.

68. Another acyclic analog is methadone (2.27) - as
potent as morphine, but eliminated more slowly.
(-)-isomer used in the
treatment of opioid abstinence
syndrome

69. If every cut gives lower potency, either most of
the molecule is in the pharmacophore or the cut
causes a conformational change away from the
bioactive conformation.
Can try adding groups to
increase the pharmacophore.
3200 times more potent than morphine

70. Fentanyl and derivatives are the most potent
opiates known.
N
N
O N
N
O
O
O
fentanyl
100X morphine
carfentanil
10,000X morphine

71. Functional Group Modification
Antibacterial agent carbutamide (2.29, R = NH2)
has an antidiabetic side effect.
Replacement of NH2 by CH3 gives tolbutamide
(2.29, R = CH3), which is an antidiabetic drug
with no antibacterial activity.
An obvious relationship exists between molecular
structure and its activity.

72. Structurally specific drugs (most drugs):
Act at specific sites (receptor or enzyme)
Activity/potency susceptible to small changes in
structure
Structurally nonspecific drugs:
No specific site of action
Similar activities with varied structures (various
gaseous anesthetics, sedatives, antiseptics)

73. SAR: Structure – Activity Relationship
Neighbourhood in the
descriptor space
Similar activity
values
Descriptor
Activity
The structure–activity relationship (SAR) is the relationship between
the chemical or 3D structure
of a molecule and its bio-
logical activity.

74. SAR for Paclitaxel
Paclitaxel (Taxol) - anticancer drug

75. Development of Taxol
• Taxol (paclitaxel) is produced from the bark of
Taxus brevifolia
• Taxol is probably the most significant drug
developed through the NCI-USDA program

76. Bark supply
• Yield of Taxol is ~0.5 gram per 30 pounds of bark
• Average 100 yr old Pacific Yew tree yields ~20 lb
of bark (3 trees/g)
• Usual treatment 2 g/patient (6 trees)
• 12,000 women dying yearly from ovarian cancer -
24,000 g of taxol - 72,000 trees
• Meanwhile significant activity shown in metastatic
breast cancer - 40,000 deaths per year

77. Supply remains a problem
• Concern – not enough trees to treat patients
• Survey by Forest Service and Bureau of
Land Management (funded by Bristol-
Myers Squibb) found >100 million trees
• Over 1.6 million pounds of bark harvested
in 1991 and again in 1992
• Need for alternative sources

78. New Sources Identified
• Other species of Taxus contain taxol even in
needles
Although yield much lower it is a renewable
resource
• Tissue cultures of bark cells promising
• Semi-synthesis in the laboratory from
precursors in needles
• Fungal pathogen on yews also synthesizes
taxol

79. Taxus baccata - English yew
• French scientists found a semi-synthetic method of
developing taxol from a molecule in needles of
Taxus baccata
Also led to the development of a second anti-cancer
compound - docetaxel (Taxotere)
• In 1992 – Holton, FSU scientist, found an easier
semi-synthesis method – this became the method
for commercial development of Taxol
• Dec 1993 – Holton achieved total synthesis

80. NH
O
O
O
OH
AcO O OH
O
O
H
OH AcO
O
3'
2'
13
10
2
9
7
4
Reduction
improves
activity
slightly May be esterified,
epimerized or
removed without
significant loss of
activity
Oxetane ring or
small ring analog
required for
activity
Removal of acetoxyl group
reduces activity; some acyl
analogs have improved activity
Acyloxyl group essential; certain
substituted benzoyl groups and other
acyl groups have improved activity
Removal of
1-hydroxyl
group reduces
activity slightly
Free 2'-hydroxyl group,
or a hydrolyzable ester
thereof required
Phenyl group
or a close
analog required
N-Acyl group
required
Acetyl or acetoxyl
group may be
removed without
significant loss of
activity. Some acyl
analogs have
multi-drug resistance-
reversing activity
Use of a molecular activity map-
structural drawing of a lead annotated
to show where structural changes affect activity or potency
SAR Conclusions

81. DNA Extraction
Methods

83. 1. Extraction methods, When do we use
which method?
2. What to think about (inhibitors,
concentrations etc).
3. How does downstream assay influence
choice of extraction?

84. The selection of the most suitable method for sample preparati
on depends on the type of sample and the purpose of the mole
cular analysis.
There is no universal approach that would equally suit all
sample matrices and/or applications.

85. Limitations to PCR include inhibitors in foods that can
result in false positives.
Food-derived PCR inhibitors include Ca2+, fats, glycoge
n, and phenolic compounds. The presence of proteases i
n cheese and milk may also inhibit PCR
Detection of parasites in water and food samples is oft
en hampered by the occurrence of organic and inorgani
c substances

86. The main goals of sample preparation are:
• Concentrate the target organisms and, subsequently, the
template for the PCR
• Maximize the quality of DNA to produce a homogenous
sample for specific and sensitive enzymatic amplification.
• Eliminate possible PCR inhibitors
(i) inactivate of the Taq polymerase
(ii) degrade or capture of the nucleic acids
(iii) interfere with the cell lysis step
• Maximize DNA recovery

87. Pre-extraction concentration/isolation of
target organism
IMS Crypto and Giardia (oo)cysts
(i) Immunological: use of magnetic b
eads coated with antibodies to cap
ture parasite (quite expensive and
time-consuming)
(oo)cystsand eggs
(ii) Physical: Filtration and/or density
gradient to concentrate the target
organism and remove PCR- inhibit
ory substances of different density
.

88. (iii) Physical: Washing, homogenization a
nd centrifugation to elute parasite from v
egetable/fruits surface
(iv) Enzymatic: Meat mincing and
digestion
parasites
for su
m
ceatborne
are used
h as T
oxoplasma gondii,
Trichinella spp. and Anisakidae.
Pre-extraction concentration/isolation of
target organism

89. Extractability of genetic material from helmints eggs and protozoan (o
o)cysts in stool or environmental samples is made more complex bein
g the genetic material, to be isolated, is enclosed in very robust cell w
alls
Processing procedures are recommended prior to DNA extraction to
facilitate oocyst/cyst wall disruption and nucleic acid isolation:
• fecal samples exposed to variable number of freeze-thaw cycles
• bursts of beat-beater (Fast Prep® instrument)
• ultrasound liquid processor ‘sonicator’
These preparatory steps add significantly more cost, labor, and time
to the extraction method purification and concentration steps cause s
ome loss ofoocysts/cysts in the original fecal specimen.
Pre-extraction disruption/homogenization

90. Pre-extraction disruption/homogenization
TissueLyser LT is a small bead mill which provides fast, effectiv
e disruption of up to 12 samples at the same time. This throug
hput matches that of the QIAcube, which automates sample pr
eparation using trusted QIAGEN spin-column kits. Simultaneou
s disruption and homogenization is achieved through high-spe
ed shaking of samples in 2 ml microcentrifuge tubes with stainl
ess steel or glass beads.
The FastPrep-24™ 5G Instrument is a high-speed benchtop ho
mogenizer for the lysis of up to 24 biological samples within 4
0 seconds. Uses a unique, optimized motion to disrupt cells t
hrough the multidirectional, simultaneous beating of speciali
zed Lysing Matrix beads on the sample material.

91. Post-extraction DNA concentration
DNA-IMS: specific capture of target DNA by hybridization with
biotynilated capture oligonucleotide.
• Tissue lysis: alkaline buffer; proteinase K; 56°C Incubation
• DNA denaturation at 95°C for 5min
• Hybridization with biotynilated oligonucleotide against target
(s) sequence at 55°C
• Precipitation of DNA/Oligos by Streptavidine magnetic bead
addition

92. Post-extraction DNA concentration
Chemical concentration by Alcohol Precipitation:
• Addition of 2 volume of ice cold 100% EtOH or 1 volume of RT
Isopropanol.
• Incubation
• High sped centrifugation
• Wash with EtOH 70%
to remove salts
• Water resuspension.
Physical concentration by Inverted spin low binding cellulose
filter devices (i.e. Microcon) with high cut-off to retain DNA a
fter centrifugation.

93. DNA quantification
Spectrophotometricquantification
• Spectrophotometer equipped with a UV lamp
 Absorbance readings are performed at 260nm where DN
A absorbs light most strongly (1 OD (optical density – ab
sorbance value) is equal to 50.0 ng/ml of ds DNA)
NanoDrop
 Spectrophotometer for measurement of 0.5 – 2 µL DNA,
RNA, and protein samples. Measure very low and high c
oncentration samples (0.4 – 15,000 ng/µL dsDNA)
Fluorimetric quantification (Qubit)
 A fluorescent intercalating dye whose fluorescence is gre
atly enhanced when bound to double stranded DNA
 >sensitive, uses as little as 1 μL of sample accurate for in
itial sample concentrations from 10 pg/µL to 100 ng/µL
 Quantitation is done by comparison to a standard curve
which was constructed from standards

94. What are the Most Commonly used DNA
Extraction Procedures
• Organic (Phenol-Chloroform) Extraction!
• Non-Organic (Proteinase K and Salting out)
• Silica Based (Silica exchange resin)
• Chelex (Ion Exchange Resin) Extraction
Maximize the quality of DNA! You will
improve PCR success!!

95. Inhibitors:
Complex biological samples can contain a wi
de range of inhibitory substances which are
capable of reducing or preventing PCR ampli
fication!
Different samples can have very different co
mpositions, and the presence of substances
potentially inhibitory to the PCR can vary de
pending on the sample type and compositio
n.

96. Inhibitors:
… but also inhibitors from the DNA purification process!
•alcohols like ethanol and 2-propanol (isopropanol) from DNA
template precipitation
•organic solvents like phenol from phenol/chloroform
purification
•salts like KCl, NaCl from precipitation
•detergents like SDS,.. from membrane lysis
Therefore, the selection and evaluation of the sample preparatio
n approach and a suitable reaction mixture, including polymeras
es and primers, are critical to obtain PCR-compatible samples of
comparable composition, irrespective of the variation in the origi
nal matrix (e.g. batch-to-batch variation)
Do not forget to turn on the machine!!!

97. How prevent or lower the amount of PCR inhibition!
 sample processing and/or decreasing the amount of sample
matrix, thereby removing or diluting matrix-derived inhibitors
 Favor extraction methods that are proven to eliminate
inhibitors from the purified template DNA
 adding different components
to the PCR reaction
 The choice of DNA polymerase can have a large impact on
resistance to inhibition (Inhibitor-resistant chemistries!)

98. Why to use a commercial kits?
Available for specific types of matrices (blood, soil, culture,
food and feed)
Non-toxic methods
Short incubation and centrifugation times
Easy procedure steps
Able to get rid of inhibitors that are solvable in the aqueous
phase
Ready for automatisation

99. Why to use a commercial kits?
The commercial kits can be divided into 2 groups
Manual purification
Automatic systems
•Most of the manual kits are based on solid-phase purification
normally performed using a spin column
•many of the automatic systems uses magnetic beads to capture
the nucleic acids

100. All nucleic acids extraction procedures
comprise 3 steps:
1. cell lysis (chemical, mechanical or enzymatic)
2. removal of cell contaminants
3. precipitation and purification of the nucleic acids

101. Silica matrix
Lysis of the cells:
•chaotropic agents (like guanidine thiocyanate or urea): Destabilize hydr
ogen bonds, hydrophobic interactions, proteins (including nucleases). D
isrupts the association of nucleic acids with water in preparation to bin
d to silica membrane
•Detergent+proteinase K+incubation in 56°C: For protein lysis and solu
bisation)
Purification:
•DNA binds to the silica matrix (EtOH enhances DNA binding)
•Contaminants are eliminated by washing steps
•Extraction:
•When elution buffer is added the nucleic acids become hydrated and
will release from the membrane.
•DNA more stable at a slightly basic pH and will dissolve more easily in
a basic buffer.

102. Silica-membrane-based
spin columns
•Several brand (i.e. QIAamp DNA Mini Kit; Prom
ega Wizard® Genomic) provides silica- membra
ne-based nucleic acid purification from tissues,
stool, plant, soil!
•Reduces hands-on preparation time (<30 min)
•Favor PCR inhibitors removal by adsorption
resin (i.e.InhibitEX, etc.)
•Purification can be automated (QIAcube).

103. •Automated processing of the Qiagen
spin columns
•Capacity: up to 12 samples per run
check
liquid
•Optical & Ultrasonic sensors:
samples, consumables and
loading for each protocol run
Qiacube

104. Silica magnetic bead/particle
•Use magnetic beads or particles functionalized with silica surfaces to
allow selective binding of DNA in the presence of high concentrations
of salt
•nucleic acid purification from tissues, stool, plant, soil!
•This methods also employ a bind-wash-elute process
•DNA bound to a magnetic bead can be easily separated from the
aqueous phase using a magnet
•Larger surface area for DNA to bind to
•Removes spin column clogging problem
•Sealed reagent cartridge and plasticwear reduce human
handling

105. Silica magnetic bead/particle
of high
•Faster processing and fine control of solution volumes.
•Magnetic-based methods are ideal for automation
throughput processing (no need for centrifugation)
•Very expensiveAs previously mentioned, automation limits the a
mount of alterations and optimisations you may want to do wh
en samples require more work

106. The BioSprint 96 provides automated
DNA purification of 96 samples in one
hour using MagAttract technology.

107. Spin columns are convenient and easy to use and are often the
choice of labs with lower-throughput needs.
For labs that handle or process multiple samples at once, extra
ction kits that use silica-coated magnetic beads are an option.

108. Chelex Extraction
Chelex 100, Molecular Biology Grade resin from BioRad is a highly p
ure, nuclease and ligase inhibitor-free chelating resin, certified not
to interfere with downstream PCR.

109. Chelex Extraction
Chelex 100 is an ion exchange resin that is added as a 5% solution
(wt/vol).
Chelex is composed of styrene divinylbenzene copolymers containi
ng paired iminodiacetate ions that act as chelating groups in bindin
g polyvalent metal ions such as magnesium (Mg2+).
By removing the Mg2+ from the reaction, nucleases are inactivated
and the DNA is protected.

110. Chelex Extraction
A 5% solution of Chelex is added to sample and incubated at 56°C
for 30 minutes to lyse cells and remove contaminants/inhibitors.
DNA is denatured and membranes/cellular proteins destroyed
heating sample at 100°C .
The tube containing the Chelex is centrifuged, the resin is pelleted,
the supernatant containing the DNA is removed.
Relatively fast
Minimizes contamination – uses only a single tube
Removes PCR inhibitors
Chelex extractions can be prone to degradation, and should be kept
in the freezer when not in use, or these extractions should be used wi
thin 24 hours of extraction.
Pigments or other inhibitors cannot be efficiently removed

111. Organic (Phenol/Chloroform)
Lysis of the cells:
alkaline buffer or chaotropic agents, proteinase K,
incubation in 56ºC
Purification: phen
ol:chloroform
Extraction:
ethanol or isopropanol

112. Phenol/Chloroform
aqueous sample
oPhenol Chloroform (~1:1) is added to your
containing the required DNA as well as proteins
oPhenol and water are immiscible so two phases form. Phenol is
more dense sinks to the bottom
oThe two phases mixing cause proteins to denature and partition
into the less polar organic phenol phase.
oThe sample is centrifuged
oPolar DNA remains in the polar aqueous phase whilst proteins
remain in the less polar organic phase.
oThe DNA in the solution can now be precipitated, washed and
resuspended

113. Phenol/Chloroform
•yields relatively pure, high molecular weight DNA
•Ideal for NGS application
•T
o be applied on purified parasites!!!
•Time consuming
tubes –
•Requires sample to be transferred to multiple
increases risk of contamination
•Involves use of hazardous (and smelly!) chemicals

114. How Much DNA do I need?
• It depend on the detection limit of the downstream assay
PCR<nPCR<qPCR<WGA
• The target sequence (single copy vs multicopy)
• The primer set!
How Much DNA Can I Recover?
• T
oxoplasma oocyst (8 sporozoite=8n) 0.6 pg
• Giardia cyst (16n) 0.3 pg DNA
• Cryptosporidium oocyst (4n) 0.03 pg
• Trichinella muscle larva (2n) 150 pg

115. Purified G. duodenalis cysts
4 pre-cyst treatments:
• No treatment
• Boiling (100°C/5 min) and freezing (-70°C/5 min) repeated 5×
• Boiling (100°C/2 min) and freezing in liquid nitrogen (2 min) repeated 3×
• Sonication (3×2 min)
All followed by on digestion with PK (56°C)
3 DNA extraction kits compared:
• QIAamp DNA Stool Kit (QIAGEN )
• QIAamp DNA Tissue Mini Kit (QIAGEN )
• FastDNA SPIN Kit for Soil (MP Biomedicals) without beat-beating step!
2 Detection methods
• Semi-nested PCR (β-giardin gene)
• qPCR (small subunit rRNA gene)
Best result QIAamp DNA Tissue Mini Kit preceded with triple liquid
nitrogen/water bath
LOD 50 cysts by smPCR vs 10 cyst by qPCR

116. 3 DNA extraction Methods
• QIAamp stool spin-column kit +95 °C for 10 min
• Freeze/thaw 5 cycles+QIAamp spin-column stool kit+95
°C for 10 min
• bead beating with guanidine thiocyanate using a Fa
stPrep-28 machine followed by liquid-phase silica p
urification of DNA.
Detection method
• qPCR (tpi gene)
DNA=1000cysts/g stool detectable and typable
Method 3 higher DNA yields but more susceptible to PCR
inhibitors (liquid silica matrix vs solid-state silica-based co
lumn)
A weighted, multi-attribute approach to
compare direct extraction of DNA from
15 Giardia microscopy-positive stools

117. Stool spiked with Toxoplasma or Hammondia
oocysts
200mg stool spiked with 104; 103; 102; 10; negative
Methods (kits for difficult sample)
• PSP Spin Stool Stratec
• InnuPrep Stool Analytical Jena
• NucleoSpin Soil Machenery-Nagel*
• EZNATM Stool Omega
• ZR Faecal Zymo*
• Sedimentatio-Flotation+Freeze/thaw+Phenol-
Chlorophorm
Single step PCR
The in house methos is allow alarge amount
of material to be tested! Time consuming!!
!!
Kit only allow 200-500mg of sample!
*Protocol with beat beating are more
efficient!!!!!

118. Trichinella larvae obtained by artificial digestion and preserved in
90% ethanol
Trichinella
For best results larvae must be quickly removed from the digestion fluid and
transfered in the tube filled with ethanol
Larvae in 90% ethanol can be stored at room temperature, at +4°C or at -20°C up to 5
years
Do not fix larvae in formalin, the DNA will no more be amplified

119. DNA isolation
proteinase K–SDS
digestion and phenol–
chloroform extraction[
Silica membrane based
kit
Magnetic beads based kit
(EURLP protocol)
PRO - Cheap
-Can be used on single
larvae
- Good DNA recovery
rate
- Good DNA
purification
- Excellent DNA recovery rate
- Excellent DNA purification
-Can be used on single
larvae
CON - Low DNA recovery
rate
-Non-purified DNA
(pK incubation)
- Quite expensive
-Cannot be used on
single larvae (not
enought sensitive)
- Really expensive

120. The choice of the right extraction method
depends on:
• Sample matrix/quality (isolated parasite ≠ stool sample!)
• Samples Number (10 ≠ 100!)
• Cost
• Time
• Simplicity and Experience (more steps more risk of
mistakes!)
• DNA quality and yield
• Throughput
• Downstream application
• Automation
• Safety
Better to evaluate the best extraction method experimentally

121. Bioorganic & Medicinal Chemistry 12 (2004) 1817–1834
Review
Enzymes in the synthesis of bioactive compounds:
the prodigious decades
Eduardo Garcı´a-Junceda,* Juan Francisco Garcı´a-Garcı´a, Agatha Bastida and
Alfonso Ferna´ ndez-Mayoralas
Departamento de Quı´mica Orga´nica Biolo´gica, Instituto de Quı´mica Orga´nica General, CSIC, C/ Juan de la Cierva 3.
Madrid 28006, Spain
Received 29 October 2003; accepted 16 January 2004
Abstract—The growing demand for enantiomerically pure pharmaceuticals has impelled research on enzymes as catalysts for
asymmetric synthetic transformations. However, the use of enzymes for this purpose was rather limited until the discovery that
enzymes can work in organic solvents. Since the advent of the PCR the number of available enzymes has been growing rapidly and
the tailor-made biocatalysts are becoming a reality. Thus, it has been possible the use of enzymes for the synthesis of new innovative
medicines such as carbohydrates and their incorporation to modern methods for drug development, such as combinatorial chem-
istry. Finally, the genomic research is allowing the manipulation of whole genomes opening the door to the combinatorial bio-
synthesis of compounds. In this review, our intention is to highlight the main landmarks that have led to transfer the chemical
efficiency shown by the enzymes in the cell to the synthesis of bioactive molecules in the lab during the last 20 years.
Ⓒ 2004 Elsevier Ltd. All rights reserved.
Contents
1. Introduction................................................................................................................. ........................................ 1818
2. General aspects of biocatalysis ............................................................................................................................1818
2.1. Enzymes in non-aqueous solvents................................................................................................................ 1818
2.2. Enzyme immobilization ............................................................................................................................... 1819
2.3. From enzyme over-expression to tailor-made biocatalysts ..........................................................................1819
2.3.1. The PCR revolution and the biocatalysis ......................................................................................... 1819
2.3.2. Catalytic antibodies: tailor-made biocatalysts................................................................................... 1819
3. Enzymes in the synthesis of chiral drugs .............................................................................................................1821
4. Enzymatic synthesis of carbohydrates: new tools for new drugs.........................................................................1822
4.1. Aldolase enzymes for C–C bond formation................................................................................................. 1822
4.2. From glycosidases to glycosynthases ........................................................................................................... 1823
4.3. Glycosyltransferases: one enzyme-one linkage............................................................................................. 1823
5. Enzymes enter the field of combinatorial chemistry ............................................................................................ 1824
5.1. Enzymes in polymer-supported synthesis .................................................................................................... 1824
5.2. Combinatorial biocatalysis .......................................................................................................................... 1826
3. Let the cell make the chemistry: from metabolic engineering to combinatorial biosynthesis ......................1826
1.The cell factory: an alternative strategy for the synthesis of oligosaccharides..................................1827
5.3.2. Combinatorial biosynthesis: creating new polyketides...................................................................... 1827
6. Conclusions.......................................................................................................................................................... 1830
Acknowledgements............................................................................................................. ......................................1830
References and notes ................................................................................................................................................ 1830
* Corresponding author. Tel.: +34-91-562-29-00; fax: +34-91-564-48-53; e-mail: iqogj78@fresno.csic.es
0968-0896/$ - see front matter Ⓒ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.032

122. ‘...I can forsee a time in which physiological chemistry
will not only make greater use of natural enzymes but will
actually resort to creating synthetic ones’.
Emil Fischer, Nobel Lecture, 1902.
1. Introduction
The enzymes advantages and disadvantages as catalysts
for organic synthesis, derived from their own nature.
Enzymes catalyze reactions with remarkable rate accel-
erations over background (kcat/kuncat can reach 1017).
They are chemoselective and regio- and stereospecific;
however, they usually present narrow substrate specifi-
city limiting their application. Enzymes are environ-
mental friendly catalysts since they work in aqueous
solutions and at moderate temperatures, but their poor
stability under in vitro reaction conditions can become a
strong limitation to their use in chemical synthesis.
Many enzymes work under similar conditions of pH,
temperature, etc., allowing their combination to per-
form several synthetic steps in one-pot. In this manner,
different problems related to the availability of sub-
strates or product inhibitions may be overcome.
Besides the unique properties of enzymes as catalysts,
synthetic chemists have been reluctant to employ them
for a number of reasons:
(i) most organic compounds are water-insoluble,
and the water removal is tedious and expensive;
(ii) limited availability of biocatalysts with the
desired activity and substrate specificity;
poor stability of the enzymes, and
relatively high cost of the biocatalyst.
(iii)
(iv)
These drawbacks have been, at least partially, solved by
the advances produced in the biocatalysis field over the
last 20 years. Thus, the finding that most enzymes can
work in organic solvents,1 have heightened the use of
enzymes in organic synthesis. Recombinant DNA tech-
nologies and specially the PCR,2 have facilitated
obtaining new enzymes by a variety of approaches.
Protein engineering by rational design3 or by in vitro
evolution4 permits the modification of the enzyme sub-
strate specificity, stability and other catalytic properties.
Using the diversity offered by the immunological sys-
tem, it has been possible to create tailor-made enzyme
active sites into antibodies by immunization with
appropriate transition-state analogues.5 The optimiza-
tion of recombinant expression systems allows the
application of efficient strategies for achieving high-level
expression of both natural and engineered enzymes in
different host cells, cutting down the cost of the bioca-
talyst6 and expanding the array of reactions that can be
afforded enzymatically. Finally, the use of immobilized
enzymes present two main benefits (i) easy separation of
enzyme from the reaction mixture; and (ii) the biocata-
lyst can be reused several times.7
Several excellent comprehensive reviews on enzymes in
organic synthesis have been published in recent years.8
In this review we will summarize the main landmarks
that have led to the recognition of enzymes as very use-
ful catalyst in organic synthesis and especially in phar-
maceutical processes where chirality is a key factor in
the efficacy of many drugs and working with multi-
functional molecules is not unusual.
2. General aspects of biocatalysis
2.1. Enzymes in non-aqueous solvents
For a long time enzymes were believed to work effi-
ciently only in aqueous solutions. Consequently, their
utilization in organic synthesis was rather scarce since
the low water solubility of many substrates represented
a serious obstacle. This disadvantage, nevertheless, sti-
mulated the search for systems based on the use of
organic solvents in order to increase the solubility
of hydrophobic substrates.9 The methods adopted
evolved from the mixtures of water and water-miscible
organic solvents, biphasic aqueous-organic systems,
reverse micellar systems, and finally in nearly anhydrous
organic systems. Because the latter is attractive, it has
undergone a rapid expansion among the synthetic che-
mists during the last two decades.10
In principle the replacement of water by an organic
solvent should be adverse for the catalytic function of
the enzyme, since water molecules are necessary to
maintain the catalytically active conformation of the
enzyme. However, the real question, as placed by Kli-
banov who did a leading research in the subject, should
not be whether water is required but how much of it is
necessary.1,11 As long as the hydration shell required for
retention of enzyme catalytic activity is preserved, the
replacement of the rest of the water with an organic
solvent should be possible without losing the active
conformation. In general, the catalytic activity of
enzymes in neat organic solvents is lower than in water.
But this decrease in activity could be avoided and effec-
tive remedies are emerging.12 For example, the enzyme
activity is usually higher in hydrophobic solvents than
in the more hydrophilic ones, since the latter can strip
the essential water from the enzyme molecule.13 The pH
is one of the key factors for enzyme activity, but it has
not meaning in organic solvents. Instead, in organic
solvent media enzymes have ‘pH memory’.14 In practice
this ‘pH memory’ can be achieved by dissolving the
enzyme in water of optimal pH followed by freeze-dry-
ing or solvent precipitation prior to its use in an organic
solvent. In this way the ionization state of the ionogenic
groups of the enzyme are retained in the solid state and
in organic solvent, which is also necessary for enzyme to
function.
However, it has become important to know the effects
of the surroundings of the enzyme molecules, in parti-
cular counter-ions and the structure of the solid catalyst
particles, on the activity and selectivity of enzymes in
low-water media.15
Ionic liquids are a new class of non-aqueous solvents
with non-molecular, ionic character. These solvents are
1818 E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834

123. salts that are liquid at room temperature. Through the
choice of cations and anions, the physical and chemical
properties can be optimized for each application. The
replacement of organic solvents by ionic liquids can lead
to remarkable improvements regarding reactivity, selec-
tivity and stability of the enzyme.16 For lipase catalyzed
kinetic resolution of a racemate, it has been shown, that
in most cases ionic liquids increase the enantioselectivity
of the enzyme and improved conversion compared to
organic media.17
2.2. Enzyme immobilization
The use of immobilized enzymes presents a number
of technological advantages such as the possibility of
reusing the biocatalyst and its easy separation from the
reaction mixture. There exists a great variety of immo-
bilization methods that can be grouped in several cate-
gories:7,18 (i) immobilization by entrapment; (ii) non-
covalent binding by adsorption on inert supports or by
affinity immobilization; (iii) enzyme crystallization; (iv)
covalent binding onto prefabricated carrier materials.
Sometimes enzyme immobilization is used as synonym
of enzyme stabilization. However, not all the immobili-
zation methods improved the stability of the enzyme.19
Since enzyme immobilization means the restriction of
the mobility of the enzyme, protein-protein interactions
are greatly retarded which diminishes inactivation
caused by aggregation, adsorption on the surface of
reaction vessels, dissociation of oligomeric proteins into
subunits, and bimolecular processes of proteolysis. For
a true stabilization of the enzyme it is necessary stabilize
the three-dimensional structure of the protein. This
effect is only possible to achieve with those immobiliza-
tion methods that increase the rigidity of the protein.20
3.From enzyme over-expression to tailor-made bioca-
talysts
1.The PCR revolution and the biocatalysis. Much of the
early work on biocatalysis focused on the use of
readily available enzymes, such as esterases, proteases
and lipases. Although these enzymes are still now
largely being used for the asymmetric synthesis of
pharmaceuticals, since the advent of PCR and the high-
throughput technologies, the advances in the recombi-
nant expression systems, and the genomic research, the
array of enzymes available for organic synthesis is
greatly expanding. This has made possible to carry out
enzymatic reactions that otherwise are difficult to per-
form by chemical methods, like the synthesis or modifi-
cation of complex molecules such as carbohydrates,
asymmetric carbon–carbon bond formation, kinetic
resolution of racemic epoxides or dioxigenation of aro-
matic compounds.
The natural biodiversity is one of the main sources of
new enzymes. Microorganisms living under extreme
conditions of temperature, pH, salt concentration or
pressure that are difficult or even impossible to be cul-
tured, provide a large number of new enzymes. The new
DNA techniques make possible to express enzymes
from these organisms in others that can be cultured
under controlled conditions such as Escherichia coli.
Therefore, extremophilic microorganisms are providing
biocatalysts able to work under conditions similar to
those needed in organic synthesis.21
Another source of new enzymes facilitated by the
advances in recombinant DNA technologies, is the rede-
sign of pre-existing enzymes. Like the drug discovery
process, the redesign of an enzyme can be afforded by
rational or by non rational techniques. Rational redesign
is usually restricted to those enzymes, whose structures
and mechanisms are well known and understood. The
improperly named non-rational methods are of general
application since no previous information about the
structure or mechanism of the enzyme is necessary.
These methods try to reproduce in a lab-time scale the
Darwinian scheme of natural evolution, consisting
basically in: (i) random genetic mutation; (ii) gene
recombination and (iii) selection for higher fitness
variants.
Random and exhaustive mutagenesis can be achieved
by several methods the most popular being the error-
prone PCR.22 Nature uses gene recombination to
increase the variability produced by mutation. The first
method for in vitro recombination of homologous genes
was the DNA shuffling described by Stemmer (Fig. 1).23
Other methods developed for in vitro recombination
include incremental truncation,24 staggered extension
process25 and random-priming recombination.26 The
last step for the in vitro enzyme evolution, and the real
bottleneck of this approach, is the identification of the
evolved gene with the desired characteristics. The diffi-
culty to find the best mutant in a library of 104–107
mutants is evident. The identification can be accom-
plished by screening or by selection.27 Genetic selection
strategies can be applied when enzyme activity is essen-
tial for viability and growth. Screening requires to assay
individually all the members of the library. Recent
improvements in high-throughput screening allow to
accomplish this task.28
In vitro enzyme evolution has been successfully used to
modify enzyme features that are of interest in organic
synthesis, such as substrate specificity,29 optimum pH,30
stereospecificity31 or enantioselectivity;32 this approach
has also been applied to improve enzyme stability at
high temperature33 or in organic solvents.34 A compre-
hensive list of evolved enzymes can be found in some
recent reviews.35
2.3.2. Catalytic antibodies: tailor-made biocatalysts.
Besides the great diversity of enzymes, there are many
important organic transformations for which do not
exist any biocatalyst. For this reason, an old longing of
the chemist is to create tailored biocatalysts. Basically
speaking, enzyme catalysis is based on the stabilization
of the transition-state. Thus, if the essential chemical
and structural details of enzymatic catalysis are known,
one should be able to design and build novel enzymes
from scratch. Against the generally accepted concept
that proteins with high affinity binding of stable ligands
E. Garcı´a-Junceda et al. / Bioorg. Med. Chem. 12 (2004) 1817–1834 1819

124. do not express catalytic activity, Jencks suggested at the
end of the 1960s, that stable molecules resembling
the transition state of a reaction might be used as hap-
tens to elicit antibodies with tailored catalytic activities
and selectivities.36 Since the first reports by the groups
of Lerner37 and Schultz38 in the middle 1980s, more
than 100 reactions have now been successfully accelerated
using catalytic antibodies (abzymes), including pericyclic
processes, group transfer reactions, additions and elim-
inations, oxidations and reductions, aldol condensations,
and miscellaneous cofactor-dependent transformations.39
However, the catalytic efficiency of most catalytic anti-
bodies generated to date is lower than that of their
enzymatic counterparts. One major advance for the eli-
citation of antibodies with higher catalytic efficiencies
has been the reactive immunization.40 Usually, haptens
are designed to mimic the geometric and electronic fea-
tures of the reaction transition state (Fig. 2).41 However,
many enzymatic reactions proceed throughout a cova-
lent complex between the enzyme and the substrate. In
the reactive immunization, a reactive hapten is designed
to promote a specific chemistry, such as the formation
of a covalent bond, in the binding pocket of the anti-
body during its induction. In this way, the selection cri-
terion is shifted from binding to catalysis. Using this
approach, highly efficient catalytic antibodies with
aldolase activity have been obtained (Fig. 3).42 The
substrate specificity of these antibodies is broader than
that observed with any natural aldolase and they are
able to catalyze a wide variety of intermolecular aldol
reactions between ketone–ketone, ketone–aldehyde,
Figure 2. The Claisen rearrangement of (—)-chorismate to form pre-
phenate. The conformationally restricted endo-oxabicyclic dicar-
boxylic acid 1 mimics the structure of the transition state and was used
as the template for generating antibodies with chorismate mutase
activity.41
Figure 1. In vitro molecular evolution process by DNA shuffling. This method involves the random fragmentation of homologous DNA sequences.
After purification of a pool of small fragments, these are reassembled into a full-length gene in a PCR-like reaction without primers. Homologous
sequences from different fragments hybridize and prime each other. Recombination occurs when a fragment from one copy of a gene primes on
another copy, causing a template switch. An additional PCR in presence of primers, typically yields a single PCR product of the correct size. Finally,
the cloning of this PCR product leads to a combinatorial library of chimeric gene sequences produced by a variable number of crossovers.
Figure 3. (A) The 1,3 diketone 1 was designed to elicit antibodies with
aldolase activity. This hapten can both trap the requisite Lys residue in
the antibody binding site (B) to then form the essential enamine
intermediate and induce the appropiate binding sites for the two
substrates. The aldehyde is represented by the 3-phenylpropiononyl
portion of the hapten 1.42
1820 E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834

125. aldehyde–ketone and aldehyde–aldehyde, as well as
several intramolecular aldol condensations.In order to
find a highly efficient catalytic antibody, the screening
of large libraries of antibodies is necessary. Several
methods for screening antibody libraries for catalysis
instead of binding have been developed. Catalytic
enzyme-lined immunosorbent assay (catELISA)43 is,
perhaps, the most widely used. An alternative approach
is the chemical selection, in which catalytic antibodies
are selected using a mechanism-based screening reagent.
This method was demonstrated to select antibodies with
glycosidase activity.44
3. Enzymes in the synthesis of chiral drugs
Chirality plays a crucial role in nature and it is a key
factor in the efficacy of many drugs.45 During the last
two decades, the synthesis of enantiomerically pure
compounds has emerged as one of the most important
fields of organic synthesis. For this purpose enzymes
offer the possibility of performing highly stereoselective
transformations under relatively mild reaction condi-
tions avoiding the use of more extreme conditions that
could cause problems with isomerization, racemization,
epimerization, and rearrangement.
Stereoselective transformations of chiral building blocks
in the synthesis of antihypertensive, anticholesterol,
anti-Alzheimer, anti-inflammatory, and other pharma-
ceuticals have been previously reviewed.46 For achieving
these transformations different classes of enzymes have
been exploited. The most often used ones are: (i)
hydrolases (lipases, esterases proteases/amidases), for
the synthesis of esters, acid, peptides/amides; (ii) dehy-
drogenases, for the oxidation of alcohols and reduction
of carbonyls;47 (iii) mono- and dioxygenases for hydro-
xylation of unactivated carbon;48 (iv) aldolases for the
stereoselective condensation of carbonyl compounds;
and (v) oxynitrile lyase for synthesis of chiral hydroxy
nitriles.49
Lipases have been the most popular biocatalysts in the
synthesis of optically pure drugs. These enzymes are
already commercially available and work efficiently in
organic solvents. This feature makes possible not only
to solubilize substrates which are not soluble in water,
also the desired enantioselectivity can be optimized by
varying the solvent using the criteria of polarity and
hydrophobicity.50
However, when only one of the enantiomers is required,
the enzymatic resolution of racemic substrates has the
limitation that the maximum yield is 50%. There are
several ways to overcome this problem: (i) the use of
meso compounds or prochiral substrates; (ii) stereo-
conversion of the remaining enantiomer; and (iii)
dynamic kinetic resolution. In the latter case, the sub-
strate is continuously isomerized during the resolution
process. Dynamic kinetic resolution will lead to higher
enantiomeric ratios of the products, provided that the
rate of equilibration of the substrate enantiomers is
about the same as, or higher than, the rate of removal of
one enantiomer from the system.51 One can theoreti-
cally obtain 100% yield of one enantiomer. An illus-
trative example of this approach has been described by
Ba¨ ckwall et al.52 They showed that the isomerization of
1-phenylethyl alcohol (Fig. 4) by a ruthenium catalyst
and enzymatic acylation with 4-chlorophenyl acetate as
acyl donor resulted in transformation of the racemic
alcohol to enantiomerically pure acetate. In this exam-
ple the ruthenium catalyst promotes the racemization of
the alcohol substrate. Racemization involves abstrac-
tion of the a-proton to give a ketone intermediate and
subsequent readdition of hydrogens to the ketone. In
most cases reported, the reaction proceeded with >99%
ee and in good yield.
Another alternative to kinetic resolution is parallel
kinetic resolution of racemic mixtures.53 In this
approach the slower reacting enantiomer is removed by
a parallel reaction, ideally at an identical rate. Thus
avoiding the decrease in the ee of the product at con-
version values close to 50%, due to the continuous
increase of the relative concentration (and, therefore,
the relative rate of reaction) of the less reactive substrate
enantiomer. For instance, Mischitz and Faber studied
the asymmetric nucleophilic opening of ( )-3-methyl-2-
pentyloxirane (rac-1, Fig. 5) catalyzed by a crude
immobilized enzyme preparation from Rhodococcus sp.
(NOVO SP 409).54 The reaction in aqueous buffer gave
the diol (S)-2 in only 40% ee. However, when the enzy-
matic hydrolysis of ( )-1 was carried out in the pre-
Figure 4. Dynamic kinetic resolution of 1-phenylethyl alcohol.52
Figure 5. Simultaneous asymmetric hydrolysis and azidolysis of
54
( )-2-methyl-2-pentyloxirane.
E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834 1821

126. 4. Enzymatic synthesis of carbohydrates: new tools for
new drugs
Traditionally, carbohydrates were associated with
structural functions and energy storage. Development
of glycobiology and glycochemistry during the past two
decades has revealed that carbohydrates are involved in
a broad range of biological functions, mainly related to
cell recognition events.55 Many of these events occur at
the very early stage of disease development and other
signaling processes. Control of such recognition pro-
cesses has become an important target for new drug
development.
Carbohydrates have multiple hydroxyl functions of
similar chemical reactivity; for example, five mono-
saccharides can be linked together to form linear or
branched chains that can give rise to 32 million different
compounds.56 Therefore, a special knowledge of selec-
tive reactions and protecting groups are needed to
achieve successfully their synthesis and modification.
Because of that, it is in the field of carbohydrate chem-
istry where the enzymatic transformations has made the
biggest impact during the last years.
4.1. Aldolase enzymes for C–C bond formation
Aldolases have attracted the interest of organic chemists
because their ability to catalyze the formation of C–C
bonds by an aldol addition reaction between an alde-
hyde and a ketone, with a high degree of stereochemical
control. In general, they show a very strict specificity for
the donor substrate (the ketone), but tolerate a broad
range of acceptor substrates (the aldehyde).
Dihydroxyacetone phosphate (DHAP) dependent aldo-
lases produce 2-keto-3,4-dihydroxy adducts and, with
some exceptions,57 they control the configuration of the
newly formed stereogenic centers. An additional advan-
tage of these enzymes is that they are stereo-
complementary, that is, their use allows the synthesis of
the four possible diaestereoisomers for a given pair of
substrates (Fig. 6). DHAP-dependent aldolases have
shown their utility in the synthesis of carbohydrate,
carbohydrate-like structures or non-carbohydrate com-
pounds. A comprehensive description can be found in
several excellent reviews58 and references therein.
Pyruvate or phosphoenol pyruvate (PEP) dependent
aldolases yield 3-deoxy-2-keto acids. In vivo, pyruvate-
dependent aldolases have a catabolic function mean-
while the PEP-dependent aldolases are involved in the
biosynthesis of keto acids. These aldolases provided
adducts that are not only densely but also differentially
functionalized.59 Thus, four different state of carbon
oxidation can be found in four contiguous carbons.
This substitution pattern allows transformation of
aldol adducts to a variety of complex natural products
(Fig. 7).
2-deoxyribose-5-phosphate aldolase (DERA) catalyzes
the aldolic condensation between acetaldehyde and D-
glyceraldehyde-3-phospate and determines the S con-
figuration of the newly formed stereogenic center.60
Beside acetaldehyde this enzyme is able to accept pro-
panal, acetone and fluoroacetone as donor sub-
strates.60b DERA is the only aldolase able to accept two
aldehydes as substrates, allowing sequential bi- or tri-
substrate aldol reactions.61 The unnaturall pyranose
obtained after DERA-catalyzed addition of acetalde-
hyde as donor are useful synthons for the synthesis of
natural products as it has been illustrated by the concise
total synthesis of epothilones A and C.62
Glycine dependent aldolases produce b-hydroxy-a-
amino acids that are medicines by themselves or can be
used as chiral building blocks for the production of
drugs.63 Their use in synthesis is hampered because the
poor stereoselectivity that often leads to mixtures of
erythro and threo compounds.63 Despite of this limi-
tations, L-threonine aldolase and D-threonine aldolase
have been used for the synthesis of several b-hydroxy-a-
1822 E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834
sence of the non-natural nucleophile azide, the (S)-diol,
2, and (R)-azidoalcohol, 3, were obtained in >90% and
60% ee respectively (Fig. 5). Therefore, a simultaneous
and opposite enantio-discrimination of two nucleophiles
(water and azide) was observed. This parallel kinetic
resolution afforded the (S)-diol product in higher ee.
Figure 6. The four DHAP-dependent aldolases are stereo-
complementary, allowing the synthesis of the four possible diaester-
eoisomers for a given pair of substrates.
Figure 7. PEP-dependent aldolases provide adducts with four different
state of carbon oxidation, allowing their transformation to a variety of
complex natural products.59

127. amino acids on a preparative scale.64 L-Threonine
aldolase has been also used for the synthesis of potent
sialyl Lex mimetics65 and the immunosuppressant
mycestericin D.66
4.2. From glycosidases to glycosynthases
Glycosidases are readly available enzymes and use sim-
ple glycosyl donors, that can be even the free mono-
sacharide. These hydrolytic enzymes are able to catalyze
the formation of glycosidic bonds in a stereospecific
manner.67 However, when applied to the synthesis of di-
and oligosaccharides the problem of the regioselectivity
has to be faced. In contrast to lipase and protease that
work in anhydrous organic solvents, glycosidases do
not. Therefore, when using for synthesis of glycosides
the hydrolysis is always a competitive reaction.
Several tricks have been applied to improve yield and to
control the regioselectivity of glycosidase-catalyzed
reactions:68
(ii) Use of very reactive glycosyl donors, in such a
way that it is cleaved more rapidly than the
product formed. Under these conditions, the
reaction is kinetically controlled.
Introduction of subtituents at certain positions in
the sugar acceptor. For instance, it has been
shown that both the nature of the substituent and
the anomeric configuration influence the regios-
electivity and the yield of the glycosidation.69
Manipulation of the medium, using organic
cosolvents to reduce the total volume of water.
Also, addition of salts at high concentration has
been used in order to reduce the water activity.
The use of lipid-coated glycosidases improves the
behavior of the enzyme in media with low water
activity.70
(ii)
(iii)
An alternative approach is the use of specifically muta-
ted glycosidase (named glycosynthase), which can effi-
ciently synthesize oligosaccharides, but does not
hydrolyze them.71 For the case of glycosynthases, an
active carboxylate nucleophile in the catalytic center of
the enzyme is replaced with a nonnucleophilic amino
acid side chain, resulting in an enzyme which is cataly-
tically inactive since it cannot form the glycosyl-enzyme
intermediate (Fig. 8). However, the mutant enzyme can
transfer an activated glycosyl derivative, bound at the
active site in the place of the normal glycosyl-enzyme
intermediate, to a suitable acceptor bound in the agly-
con pocket. Glycosyl-fluorides72 are appropriate acti-
vated glycosyl donors since they are readily synthesized
and have a small leaving group. As the disaccharide
products are not hydrolyzed this method provide a
high-yielding synthetic procedure of oligosaccharides. A
similar approach has been used to obtain glyco-
synthases from endo-glucanases.73
4.3. Glycosyltransferases: one enzyme-one linkage
Glycosyltransferases in vivo are responsible for the final
‘decoration’ of proteins and lipids and contributes to
the great variety and complexity of secondary metabo-
lites in plants, bacteria and other organisms. Their strict
control over the stereo- and regioselectivity of the newly
formed glycosidic bond, have led to the ‘one enzyme-
one linkage’ concept.74
The synthetic utility of glycosyltransferases has been
demonstrated in the synthesis of numerous complex
oligosaccharides and glycoconjugates.75
Enzymes have allowed affording the in vitro synthesis of
homogeneus glycoproteins, a difficult task to obtain by
in vivo methodologies since glycosylation is a post-
transcriptional modification and it is affected by several
environmental factors.76 Several strategies have been
developed in the last years for the enzymatic synthesis
of glycoproteins (Fig. 9). The combined use of endogly-
cosidases, glycosyltransferases and proteases has been
applied to the synthesis of a homogeneous RNase B
glycoform.77 The use of endoglycosidases A or M allows
the exchange of sugar chains to obtain a protein with
homogeneous N-linked glycans.78 Finally, other
approach makes use of a natural protein-splicing
mechanism mediated by inteins.79
In spite of all these impressive examples, the use of gly-
cosyltransferases in synthesis faces two majors draw-
backs: (i) their limited availability and (ii) the need of a
nucleotide activated sugar as donor.
(i) Historically, most glycosyltransferases studied
have been from mammalian sources because
these have been the focus of glycobiology and
bio-medical studies. Interest is increasing in
bacterial glycosyltransferases. Many bacterial
glycosyltransferases are able to produce mam-
malian-like structures and, in addition, in
microorganisms it is possible to find transferases
with specificities not yet found in mammalian
enzymes.80 An additional advantage of bacterial
glycosyltransferases is that they usually show
broader substrate specificity than their mammal
counterpart.81 Work in our lab on recombinant a
1,6-fucosyltransferase from Rhizobium sp.82 has
shown that this enzyme is able to accommodate a
variety of modifications on the acceptor sub-
strate. Thus, it is able to accept as substrate,
oligomers and monomer of GlcNAc and it does
not seem to present selectivity for the anomeric
configuration of the acceptor.
Figure 8. Proposed mechanism of glycosynthases. The enzyme-derived
nucleophilic carboxylate (R00 ) has been mutated to glycine, serine or
alanine.71
E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834 1823

128. in stoichiometric amount in medium or large-
scale synthesis and, even more important, the
nucleoside phosphate released during the reac-
tion is a natural inhibitor of glycosyltransferases.
This problem can be avoided by removing the
nucleotide with alkaline phosphatase83 or, in a
more sofisticated way, by in situ regeneration of
the glycosil donor. The ability of most of the
enzymes to work under similar conditions of pH
and temperature together with their high speci-
ficity for substrate, allows the combination of
several enzymes, each with different catalytic
activities, in the same reaction flask. These
properties are the basis of the strategy developed
by Whitesides et al. in 198284 for the synthesis of
N-acetyllactosamine with in situ regeneration of
UDP-Gal. Afterwards, other recycling systems
for several sugar-nucleotide have been devel-
oped.85 The use of bifuctional fused enzymes86
and ‘superbeads’ containing the necessary
enzymes for the regeneration of the sugar-
nucleotide co-immobilized on Ni-agarose,87 are
recent improvements to the traditional scheme
for in situ sugar-nucleotide regeneration cycle.
In an analogue way, multienzyme systems have
been developed for regeneration of other cofac-
tors. Because the interest of bioactive oligo-
saccharides sulfates, the methods for the
recycling of 30-phosphoadenosine 50-phospho-
sulfate (PAPS), are noteworthy.88
In the early 1990s, combinatorial chemistry revolution-
ized the drug discovery field changing the traditional
rational drug design approach for the idea of covering a
vast range of chemical diversity where a ‘hit’ could be
found faster.89 A more recent approach in combinator-
ial chemistry is to create smaller and focuses libraries.
Thus, combinatorial chemistry is used to generate a
high degree of structural and chemical diversity on
initial lead compounds in order to optimized them for
binding, target specificity, bioavailability, etc. Biocata-
lysts have been recently incorporated to the array of
synthetic tools for combinatorial chemistry with special
emphasis on focuses libraries. Biocatalysts can be
employed in two different combinatorial schemes to
generate libraries of compounds: (i) combinatorial bio-
catalysis, which assembles in vitro new compounds and
derivatives using isolated enzymes and (ii) combinator-
ial biosynthesis, which shuffled or modified the genes of
natural biosynthetic pathways to produce in vivo
libraries of ‘unnatural’ natural products.
Figure 9. Three strategies for the chemo-enzymatic synthesis of homogeneous glycoproteins. (A) Use of endoglycosidases for the exchange of sugar
chains;78
(B) combined use of endoglycosidases, glycosyltransferases and proteases for the synthesis of a homogeneous glycoforms;77
(C) glycoprotein
synthesis mediated by inteins.79
(ii) The sugar-nucleotide used by glycosyl- 5. Enzymes enter the field of combinatorial chemistry
transferases as donor, is too expensive to be used
5.1. Enzymes in polymer-supported synthesis
Combinatorial chemistry often involves the synthesis on
polymeric supports. The advantage of this synthetic
methodology mainly stems from the ease work up of
reactions, which allows the automatization of the process.
In polymer-supported synthesis the substrate is
anchored to the polymer through a functional group,
1824 E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834

129. the so-called linker, which must be stable during the
synthesis and has to be cleavable at the end of the syn-
thetic route with high selectivity. Under the mild condi-
tions where enzyme works most of compounds are
stable and therefore, the use of enzymes has open up
alternative opportunities to release compounds from
polymeric supports. Enzyme-labile linkers have been
In a different approach using soluble monomethylether
of polyethyleneglycol as support, glycosidases have been
used for both glycosidation of a sugar acceptor and for
removal of the unreacted monosaccharide acceptor.96
As shown in Figure 11, the glucose anchored to the
soluble support is galactosylated using b-galactosidase.
Then, the unreacted monosaccharide glucose was
removed by the combined use of a- and b-glucosidases
to obtained only MPEG-bounded disaccharides.
Finally, disaccharides were released from the polymer
by cleavage of the linker.96a
Recently, Flitsch et al.97 reported the first example of
protease-catalyzed high-yielding peptide synthesis on
E. Garcı´a-Junceda et al. / Bioorg. Med. Chem. 12 (2004) 1817–1834 1825
developed, involving the use of hydrolases such as pro-
tease, lipase or acylase.90 For instance, Waldmann et al.
described the exo-linker 1, which comprises a 4-acyloxy-
3-carboxy-benzyloxy group (Fig. 10).91 The linker 1 is
attached as an amide to the solid phase. It contains an
acyl group, for example acetate, which can be cleaved
by lipases or estereases. Cleavage of the acyl group by a
lipase generated a phenolate 2, which fragments to give
a quinone methide 3 and releases the desired product 4.
The quinone remains attached to the solid phase and is
trapped by water or an additional nucleophile.
Enzymes can also be exploited as efficient and selective
catalyst in solid-phase synthesis where conventional
chemistry is laborious and difficult. Wong et al. repor-
ted the synthesis of sialyl Lewisx glycopeptide on a solid
support by the use of glycosyltransferases.92 In addition,
the target product was detached from the solid support
through a protease-catalyzed hydrolysis. After this
work, several syntheses of oligosacharides on solid sup-
port93 or on soluble polymeric support94 have been
reported. Solid-phase and liquid-phase synthesis of
glycopeptides, glycolipids, oligonucleotides and oligo-
saccharides have been reviewed by Zehavi.95
Figure 10. Principle for the development of the enzyme-labile 4-acyl-
oxybenzyloxy linker group.91
Figure 11. General strategy for the liquid-phase synthesis of disaccharides using glycosidases.96a

130. solid support in bulk aqueous buffer, with no need for
organic cosolvent or activated carboxylic acid.
These and other many examples of solid and liquid-
phase biocatalytic synthesis show that the biocatalytic
synthetic machinery is ready to be used for the pre-
paration of compound libraries.
5.2. Combinatorial biocatalysis
The creation of focused libraries is based on derivatiza-
tion of existing molecules. This approach mimics the
chemistry that occurs in biological systems where pre-
cursors are modified by the action of biocatalysts. For
modification of existing lead compounds, the special
properties of enzyme as catalysts report some advan-
tages over synthetic chemical reactions:98
(ii)
(iii)
(i) the high chemo-selectivity of enzymatic reactions
allows modifying only one kind of functional
groups in a scaffold molecule that typically con-
tains several different functional groups.
the enzyme regioselectivity provide the opportu-
nity for specific combinatorial modification of
lead molecules with multiple copies of the same
functional group, for example, glycosylation
catalyzed by glycosyltransferases with different
regioselectivity.
the enzyme stereocontrol over the reaction permit
a combinatorial approach to the three-dimen-
sional structure of the compound, for example,
using the four DHAP-dependent aldolases.58b
A myriad of biocatalytic reactions are available for
combinatorial biocatalysis (Table 1). These reactions
can be grouped in three mayor categories:98a (i) intro-
duction of new functional groups; (ii) modification of
existing functionalities; and (iii) addition onto func-
tional groups.
An important issue regarding combinatorial bicatalysis
is that of orthogonality.99 Modification of a substrate
by one enzyme, ‘A’, may prevent it from being sub-
strate for another enzyme, ‘B’, while the modification of
the initial substrate by ‘B’, may not preclude it from
being a substrate for the first enzyme, ‘A’ (Fig. 12). The
reactions are performed iteratively. A first generation of
derivatives is modified by another round of biocatalytic
reactions at additional reactive sites to produce a second
generation of derivatives. After several iterations is
possible to create a great number of derivatives from the
original lead compound (Fig. 13).
Although combinatorial biocatalysis is an emerging
technology in the field of drug discovery, published
application have expanded at a growing rate. New
advances including iterative derivatization of small
molecules and complex natural products, regioselec-
tively controlled libraries, novel one-pot library synth-
esis, etc., have been recently reviewed.100
5.3. Let the cell make the chemistry: from metabolic
engineering to combinatorial biosynthesis
Fermentation processes is a traditional approach of the
pharmaceutical industry for obtaining natural-bioactive
products. The possibility to manipulate metabolic path-
ways at genetic level has opened the door to use the cell
like a chemical factory for the production of new
‘unnatural’ natural products. The whole-cell approach
Introduction of new functional groups Modification of existing functional groups Addition onto functional groups
Carbon–carbon bond formation Oxidation of alcohols to aldehydes and ketones Acylation
Hydroxylation Reduction of aldehydes and ketones to alcohols vynil esters
Hydrogenation Oxidation of sulfides to sulfoxides trihaloethyl esters
Halogenation Oxidation of amino groups to nitro groups vinyl carbonates
Peroxidation Oxidation of thiols to thioaldehydes vinyl carbamates
Epoxidation Hydrolysis of nitriles to amides and carboxilic acids oxime esters
Cycloadditions Replacements of amino groups with hydroxyl groups oxime carbonates
Halohydrin formation Lactonization bifunctional esters
Addition of amines Isomerization Glycosylation
Epimerization glycosides
Dealkylation aminoglycosides
Methyl transfer glycosilic acids
Amidation
amides
peptides
hydrazides
Phosphorylation
phosphates
phospholipids
1826 E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834
Table 1. Enzyme catalyzed reactions with application to combinatorial biocatalysis98a
Figure 12. Orthogonality of biocatalysts.99

131. is especially appealing to obtain compounds produced
by the consecutive action of several enzymes or when
co-factor regeneration is required.
5.3.1. The cell factory: an alternative strategy for the
synthesis of oligosaccharides. Recombinant whole-cells
over-expressing glycosyltransferases, have been used as
biocatalyst for the synthesis of oligosaccharides101 in a
similar way that is done with the isolated enzymes. That
is, the sugar-nucleotide donor and the corresponding
acceptor are incubated in the presence of the recombi-
nant cells. The concept of cell factory implies the use of
the cell metabolic machinery — natural or engineered
— for the production of the desired compound from
simple and inexpensive substrates.
Thus, E. coli cells transformed with a plasmid codifying
for the enzymes to recycle UDP-Gal — sucrose synthase
and UDP-galactose-4-epimerase102 — and different
galactosyltransferases have been used as a galactoside-
producing factory.103
Endo et al. have developed an alternative approach for
the large-scale production of sugar-nucleotides and oli-
gosaccharides through coupling of engineered bac-
teria.104 This system is based in the combined use of
Corynebacterium ammoniagenes, a bacterium able to
produce UTP from inexpensive orotic acid, with E. coli
strains metabolically engineered with the different
sugar-nucleotide biosynthetic genes. Therefore, E. coli
cells expressing galactose-1-phosphate uridyltransferase,
galactokinase, glucose-1-phosphate uridyltransferase
and pyrophosphatase, coupled with C. ammoniages cells
were able to accumulate 44 g/L of UDP-Gal in the cul-
ture supernatant after a 21 h reaction (Fig. 14A).105 In a
similar way, system for the production of other sugar-
nucleotides like UDP-glucose, UDP-N-acetylglucos-
amine,106 GDP-mannose, GDP-fucose107 and CMP-
neuraminic acid108 have been developed. For this last
system, two recombinant E. coli strains were used; one
expressing the CTP synthase and the other expresses the
CMP-NeuAc synthase (Fig. 14B).
When these sugar-nucleotide production systems are
coupled with a new strain of E. coli over-expressing a
glycosyltransferase, different oligosaccharides can be
efficiently produced (Fig. 14).105,107—109
5.3.2. Combinatorial biosynthesis: creating new polyke-
tides. Polyketides belongs to a very important family of
compound for the pharmaceutical industry. Nowadays,
more than 40 polyketide drugs are in the market
Figure 13. Iterative synthesis of a 600-member library from the flavonoid bergenin.98b
E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834 1827

132. Figure 14. Production of sugar-nucleotides and oligosaccharides by combined use of C. ammoniagenes with E. coli strains metabolically engineered.
(A) Production system for UDP-Gal and globotriose.105 (B) Production system for CMP-NeuAc and 30-sialyllactose.108
Figure 15. The erythromycin PKS system. The pathway has three PKS polypeptides containing six modules and loading and releasing domains. The
cyclized structure shown needs to be further modified to bring erythromycin to its final structure.
1828 E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834

133. including antibiotics, immunosuppresants, antifungals
and cancer chemotherapeutics.110 Production of new
polyketides is probably the best example showing the
advanced application of combinatorial biosynthesis,
which has been facilitated by the modular structure of
polyketide synthases (PKSs). PKSs complex catalyzes
the assembly of specific types of polyketides from simple
carboxylic acids.111 The synthesis begins when the
starter substrate is loaded in the acyl carrier protein
(ACP) as a thiol ester. Then, the acyl moiety is trans-
ferred to b-ketoacyl:ACP synthase (KS) domain leading
to a two-carbon atoms increase of the chain length. This
is followed by successive two carbon extensions in a new
ACP. Eventually, the polyketide is completely synthe-
sized and, then, released by the activity of a specific
thioester (TE) domain (Fig. 15).
PKSs control the selection of the starter substrate, the
length of the chain, the reduction degree and the ste-
reochemistry of the newly formed chiral centres.112
Thus, inactivating, altering the order of the genes in the
PKS biosynthesis pathway or exchanging genes from
different organisms, novel hybrid polyketides can be
obtained (Fig. 16).113 However, the design of new poly-
Figure 16. Two different strategies for the synthesis of ‘unnatural’ natural products by combinatorial biosynthesis. (A) Precursor-directed polyketide
biosynthesis; inactivation of the first ketosynthase (KS) prevent the production of the second-step precursor, allowing to prime the second module
with synthetic analogues of the product of the first module.113a (B) Introduction of auxiliary catalytic domains; by replacing the ketoreductase (KR)
domain of module 2 with a KR-DH(dehydratase)-ER(enoylreductase) tridomain from the rapamycin synthase ( ) new chemistry is introduced
into the reaction sequence catalysed by the module.113c
E. Garcı´a-Junceda et al. / Bioorg. Med. Chem. 12 (2004) 1817–1834 1829

134. ketides must face some dificulties. After being released
from the PKSs, the polyketide must be cycled by non-
PKS enzymes and heterologous cyclases may not work
properly on the ‘unnatural’ linear intermediate, which
results in the formation of incorrectly cyclized pro-
ducts.114 Also, the ACPs are not always inter-
changeable.114a Nevertheless, this approach has
provided a large number of novel compounds.115
The final enzymatic trimming with carbohydrates,
methyl groups, etc., gives even more structural diversity
to the resulting aromatic compounds than the PKS. The
importance of the sugar moiety in the biological activity
of these compounds has led to the idea of using combi-
natorial biosynthesis to modify the glycosylation pat-
tern of the aglycon.116 The richest group of sugars
present on many polyketides is the 6-deoxyhexoses. A
great number of genes involved on the biosynthesis of
these carbohydrates have been already identified and
isolated.117 Many of the glycosyltransferases involved in
the biosynthesis of secondary metabolites are known to
possess relaxed specificity for the sugar moiety and the
aglycon.118
Valuable hybrid pharmacological compounds such as
antibiotics can be made by combinatorial biosynthesis
with bacterial deoxysugar biosynthesis genes. One
approach is to introduce heterologous genes into a
mutant strain blocked in the biosynthesis of its own
deoxysugar.119 These recombinant strains can synthe-
size novel sugar derivatives through the combined
action of the host genes and the incorporated genes.
Another approach to incorporate different sugars in the
aglycon is to exchange glycosyltransferase genes
between species producing structurally related bioactive
compounds.120
6. Conclusions
Medicinal chemistry has decisively contributed to the
humankind welfare during the last century. Nowadays,
the appearance of new diseases, the rising of antibiotic-
resistant pathogenic bacteria, the growing consciousness
of the variable activity of different optical isomers, the
search for higher selectivity and less toxicity, the indivi-
dualization of the medicine in the post-genomic era,
etc., are new challenges that pharmaceutical research
must face. In this new landscape, the exciting advances
in the biocatalysis field during the past years have
placed this technology in position to play an important
role in the drug discovery process.
Acknowledgements
We apologize to those authors whose research have not
been mentioned because of the space and scope limi-
tations of this review. AB was supported by a post-
doctoral I3P contract of the European Social Found.
This work was supported by the Spanish DGI (Grants
BQU2001-1503 and PTR1995-0568-OP).
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Dr. Eduardo Garcı´ a-Junceda was born in Madrid,
Spain, in 1961. He studied Biology at the Complutense
University of Madrid (Spain). He completed his doc-
toral studies in 1990 at the Plant Physiology Depart-
ment, Faculty of Biology (UCM) under the supervision
of Prof. Dr. C. Vicente Co´rdoba. From 1990 to 1992, he
worked as a postdoctoral fellow at the CSIC first in the
Department of Instrumental Analysis and Enviromental
Chemistry (group of Dr. J. Sanz) of the Institute of
Organic Chemistry and later in the Department of Bio-
catalysis (group of Dr. J. M. Guisa´ n) of the Institute
of Catalysis and Petrochemistry. In 1992 Dr. Garcı´a-
Junceda joined as postdoctoral fellow the Dr. Chi-Huey
Wong’s Group in The Scripps Research Institute (La
Jolla, California), where he began to work on the
obtainment and use of enzymes for carbohydrate
synthesis. After his return to Spain in 1995, he was
appointed Research Staff Member in the Institute of
Organic Chemistry of CSIC in 1997. Dr. Garcı´a-Junceda’s
research interest include development of new bio-
catalysts and their application in the chemo-enzymatic
synthesis of bioactive compounds, with particular
emphasis on carbohydrates and glycoconjugates.
Juan F. Garcı´ a-Garcı´ a was born in Madrid, Spain i
n
1975. He studied for his B. A. in Biochemistry at the
Auto
´noma University of Madrid, graduating in 2001. In
2001 he joined the Institute of Organic Chemistry
belonging to the Spanish Council for Scientific Research
(CSIC), where he started his D. Phil. Studies under the
supervision of Dr. Eduardo Garcı´ a-Junceda, investigat-
ing the use of several carbohydrate-related enzymes in
the synthesis of different carbohydrate compounds.
Biographies
E. Garcı´a-Junceda et al. / Bioorg. Med. Chem. 12 (2004) 1817–1834 1833

138. Dr. Agatha Bastida was born in Madrid, Spain, in 1964.
She received her degree in Chemistry at the Auto´ noma
University of Madrid. In 1992, she obtained her PhD in
the Laboratory of Dr. J. M Guisa´n at the CSIC working
on synthesis of peptides with immobilized enzymes.
From 1993 to 1994, as Postdoctoral Fellow of the Eur-
opean Union, she worked at the Research Department
of Unilever (United Kingdom) on synthesis of polyols
esters with enzymes. She returned to Spain, to the
Institute of Catalisis (CSIC), where she worked on the
resolution of racemic carboxylic acid esters and regio-
selective hydrolysis of peracetylated sugars with lipases.
In 1998 she joined Dr. Eduardo Garcı´a-Junceda’s group
where she is working on the cloning and expression of
glycosyltransferases and their use in synthesis.
Dr. Alfonso Ferna´ ndez-Mayoralas was born in Madrid,
Spain, in 1960. He studied Chemistry at the Auto´ noma
University of Madrid and received his PhD degree
from this university in 1987, under the supervision of
Professor Manuel Martı´ n-Lomas. Postdoctoral periods
with Professors Sinay¨ (ENS, Paris), Gigg (NIMR, Mill
Hill) and Klibanov (MIT) followed. In 1990 he joined
the Spanish Council for Scientific Research (CSIC)
where he currently holds the position of Scientific
Researcher. His research interests include enzymatic
synthesis, carbohydrate chemistry, bioorganic and
medicinal chemistry.
1834 E. Garcı´a-Junceda et al./ Bioorg. Med. Chem. 12 (2004) 1817–1834