Timor BAASOV: RESEARCH FOCUS
Research in our laboratory encompasses a variety of projects directed toward
development of new chemical and enzymatic strategies for the synthesis of
biologically active compounds and designed molecules as mechanistic probes for
enzymatic reactions, carbohydrate-mediated biological recognition and catalysis.
The group has a tight collaboration with specialized laboratories in the fields of
protein engineering, rapid-quench kinetics, solid-state NMR and protein
crystallography. Current research areas include:
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Rational design of novel antibacterial drugs
Design, synthesis and evaluation of catalytic oligosaccharides
Development of new synthetic methodologies, chemical and enzymetic,
for the assembly of oligosaccharides
Rational design of novel antibacterial drugs
Toward this goal, a collaborative effort is under way to understand the
structure and function of the enzyme KDO8P synthase, a new target for such drugs.
KDO8P synthase catalyzes the synthesis of a unique sugar (KDO) that is essential for
the bacterial growth and virulence, but it does not exist in the mammalian cells. This
advantage makes KDO8P synthase an attractive target for the development of new
synthetic antibiotics.
This interdisciplinary research program brings together the synthetic organic
chemistry, biochemistry, the art of protein isolation, the nature of the enzyme system,
and the intricacies of enzyme kinetics. Generally enzymes are controlling all parts of
life. A little damage in their work might cause many disorders and various diseases.
Therefore, a deeper understanding of the mechanism of the key enzymes will surely
present new opportunities for uncovering selective antibiotics, antifungal materials,
new organic materials for treatment of various diseases.
In extending the investigation into the mechanistic details of the enzymecatalyzed reaction, we provided the first experimental proof for the distinction
between the various postulated mechanisms [Biochemistry, 37 (46), 16390-16399,
(1998)], and demonstrated the validity of proximity effect as a major role of the
enzyme in the catalysis of the initial condensation step between two substrates.
During last years we particularly concentrate on structure-function studies of this
enzyme system. Having in hands the most powerful mechanism-based inhibitor
designed and synthesized in our laboratory, [Bioorganic & Medicinal Chemistry,
7(12), 2671-2682 (1999)], we provided the first crystal structures of the enzyme in its
binary complexes with this inhibitor and with the substrate PEP [Biochemistry, 40,
6326-6334, (2001)]. As a complimentary study to the protein X-ray studies, we also
provided the first direct identification of the enzyme active site residues by solid-state
REDOR NMR. [J. Am. Chem. Soc., 122 (11), 2649-2650, (2000)]. The results of these
studies were rigorously analyzed with implication to the catalytic mechanism of the
enzyme-catalyzed reaction.
Design, synthesis, and evaluation of catalytic oligosaccharides
The long-term objective of this project is to discover the hitherto unknown
natural macromolecules bearing catalytic activity, with a major focus on
polysaccharides as potential candidates for such activity. Note that, although the
structural diversity of polysaccharides identified them early on as effective carriers of
A
information, no case of catalytically active natural polysaccharide has yet been found.
We presented a pioneering hypothesis that considers polymeric carbohydrates as
ancient organic catalysts in the pre-RNA world. To test this hypothesis, we have
formulated two complementary strategies. The first strategy utilizes a screening
approach to search for catalytic activities among natural oligo- and polysaccharides.
The second one focuses on the rational and/or semi-rational approaches to design
artificial carbohydrate-based catalysts.
As such primordial catalyst, we first designed and synthesized
pentasaccharide 1 (Fig. 1) and showed that it adopts marked rate enhancement (~500fold) and specificity for the hydrolysis of GTP to GDP and orthophosphate [Organic
Letters 3, 4311-4314, (2001)]. Although the study on structure-function relationship
of GTP hydrolysis by 1 is only at the beginning, we believe that such efforts should
lead to a new era in our understanding of biocatalysis and catalysis required for
design. Future programs in this regard include: (1) Rigorous delineation of catalytic
mechanism of 1 for the observed GTPase activity; (2) Exploration of other
oligosaccharide structures for both hydrolytic and ligase activities; (3) Rational design
of polysaccharide-based self-replicating catalytic systems.
HO
HO
OH
OH
O
HO
HO
O
O
HO
MeO
CONHMe
O
CONHMe
O
O
NH3
O
HO
HO
O
NH3
O
HOHO
1
O
GTP
1
GDP + OP
pH = 7
HO
NH3 HO
Fig 1: Organic Letters 3, 4311-4314, (2001).
Development of new synthetic methodologies, chemical and enzymetic, for the
assembly of oligosaccharides
In order to facilitate the research toward the catalytic oligosaccharides, we
have developed one-pot approach to the synthesis of oligosaccharides. We have
shown that the triglucosamine part of 1 as well as its structural analogs and
tetraglucosamine (Fig. 2) can be efficiently constructed in a one-pot manner [Organic
Letters 4, 281-283, (2002)]. This has been done by determining anomeric reactivities
of different glycosyl donors and acceptors using various protecting groups on the
B
sugar ring [J. Am. Chem. Soc., 121 (4), 734-753, (1999)]. Future programs in this area
include: (1) Further development of one-pot oligosaccharide synthesis methodology
for the construction of the libraries of 1 and the related structures via a combinatorial
manner; (2) The use of engineered thermostable glycosidases (prepared in
collaboration with Prof. Shoham’s laboratory at the Department of Food Engineering
and Biotechnology, Technion) as glycosynthases for the enzymatic synthesis of
oligosaccharides.
OAc
AcO
AcO
OAc
OH
O
SEt
TrocHN
BzO
BzO
O
AcO
AcO
SPh
TrocHN
3
1
2
4
OH
BzO
BzO
OH
O
PhthN
SPh
BzO
BzO
O
PhthN
OMP
O
O
TrocHN
BzO
BzO
O
TrocHN
BzO
BzO
(63%)
O
O
PhthN
BzO
BzO
Fig. 2: One-pot synthesis of tetrasaccharide. Organic Letters 4, 281-283, (2002).
O
O
PhthN
OMP