Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Hoechst AG H26359PC
Novel substance library and supramolecular complexes prepared therewith
Description
The present invention relates to a substance library, to a process for the
preparation thereof, to a process for the preparation of supramolecular
10 complexes using this substance library and to the use of the
supramolecular complexes prepared by means of the substance library,
and to the use of the substance library itself.
Combinatorial strategies are important approaches in the search for novel
15 active substances, especially in respect of finding lead structures and
optimization thereof: there is simultaneous and usually automated
synthesis of ensembles of structurally related compounds; the mixtures
resulting thereby (called libraries) contain hundreds, thousands or even
millions of individual compounds, each in a small amount. If the activity of
20 one component in the mixture is detected by screening, the subsequent
work of the chemist is restricted to determining the identity, because, after
all, the synthesis protocol is known.
Whereas initial substance libraries were mainly of molecules with a linear
25 constitution, such as peptides [K.S. Lam, S.E. Salmon, E.M. Hersh, V.J.
Hruby, W.M. Kazmierski, R.J. Knapp, Nature 1991, 354, 82-84], interest is
now centered in particular on "small" molecules which are important in the
area of active substances, such as heterocycles [L.A. Thompson, J.A.
Ellman, Chem. Rev. 1996, 96, 555-600]. The aim is to generate molecular
30 diversity in order to speed up the finding of lead structures and optimization
thereof.
The characteristic of combinatorial chemistry hitherto is that the synthesis
takes piace under kinetic control and that the variation by synthesis is
35 separated from the selection. This relates to the in vitro evolution of RNA
aptamers [J.R. Lorsch, J.W. Szostak, Nature 1994, 371, 31-36.] just as
much as to the search for receptors using combinatorial methods [Y.
Cheng, T. Suenaga, W.C. Still, J. Am. Chem. Soc. 1996, 118, 1813-1814],
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in which case two short peptide libraries were assembled on a steroid
framework and irreversibly linked thereto.
It is an object of the present invention by providing a new type of
5 substance library to increase by orders of magnitude, compared with
substance libraries hitherto, the number of binding sites for ligands or
substrate molecules investigated for their binding properties by means of a
substance library, by reversible combination of, in each case, two or more
identical or different molecular species present in the substance library, the
10 intention being that the combination of the molecular species present in the
substance library take place only in the presence of the substrate molecule
to be investigated, via the appropriate binding interactions with the
molecular species.
15 It is another object of the present invention to provide supramolecular
complexes which arise through combination of the molecular species
present in the substance library and through the binding interactions with
the substrate molecule to be investigated.
20 Substance libraries of this type, and supramolecular complexes of this type
ought to be suitable for producing medicinal active substances, active
substances for crop protection, catalysts or for diagnosing diseases.
The object stated at the outset is achieved by a substance library
25 obtainable by coupling different or identical molecular species, which are
preferably present in a substance library, to a molecular pairing system.
It is possible by means of the substance library according to the present
invention to prepare supramolecular complexes by exposing the substance
30 library to an interaction with a substrate, identifying and, where
appropriate, isolating the supramolecular complex formed thereby.
The present invention accordingly also relates to the provision of a
supramolecular complex prepared in this way, which is suitable, for
35 example, for producing medicinal substances, active substances for crop
protection, catalysts, for diagnosing diseases and for producing
corresponding diagnostic kits.
In the same way, the precursor of these supramolecular complexes, mainly
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the substance library according to the invention, is also suitable for
producing medicinal substances, active substances for crop protection,
catalysts and for diagnosing diseases, including the production of
corresponding diagnostic kits.
The general terms mentioned above or used hereinafter for explaining the
invention and in the claims are defined below.
Molecular species: for example molecules with a linear constitution such as
10 peptides, in particular proteins, peptoids, linear oligo- or polysaccharides, nucleic acids and their analogs or, for example, monomers such as
heterocycles, in particular nitrogen heterocycles, or molecules not with a
linear constitution, such as branched oligo- or polysaccharides or else
antibodies.
Supramolecular complex: produced by association of two or more
molecular species which are held together by non-covalent forces.
Pairing systems: supramolecular systems of non-covalent interactions
20 which are characterized by selectivity, stability and reversibility and whose properties are preferably influenced thermodynamically, such as, for
example, by temperature, pH, concentration. Examples are preferably
pyranosyl-RNA, CNA, DNA, RNA, PNA.
25 Interactions are preferably hydrogen bonds, salt bridges, stacking, metal
liganding, charge-transfer complexes and hydrophobic interactions.
Substance library: ensemble of compounds of different structures,
preferably oligomeric or polymeric peptides, peptoids, saccharides, nucleic
30 acids, esters, acetals or monomers such as heterocycles, lipids, steroids.
Substrate: molecules, preferably medicinal substances and active
substances for crop protection, metabolites, physiological messengers,
derivatives of lead structures, substances which are produced, or produced
35 to an increased extent, in the human or animal body in the event of
pathological changes, transition state analogs or else peptides, in
particular proteins, peptoids, linear oligo- or polysaccharides, nucleic acids
and their analogs, or, for example, monomers such as heterocycles, in
particular nitrogen heterocycles, or molecules not with a linear constitution,
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such as branched oligo- or polysaccharides or else antibodies, and
substance libraries, also sites of action of drugs, preferably receptors,
voltage-dependent ion channels, transporters, enzymes and biosynthetic
units of microorganisms.
Transition state analogs: synthetic molecular species which are structurally
similar to the assumed transition state of a chemical reaction but are, in
contrast thereto, stable.
10 Identification can comprise isolation or characterization of the
supramolecular complex, but preferably differentiation on the basis of
particular properties of the supramolecular complex of substance libraries
coupled to pairing systems and substrate, preferably different
chromatographic, electrophoretic, spectroscopic or signal (labeling)
15 behavior by comparison with uncomplexed species or by covalent
(chemical) attachment of the species involved in the complex formation.
CNA: cyclohexylnucleooligoamide; represents a synthetic variant of the
DNA structure in which the phosphate-sugar backbone is replaced by 2-(3-
20 aminocyclohexyl)ethanoic acid units, with the units being linked together inthe manner of a peptide, and the 3-aminocyclohexyl substituents each
being provided with a nucleobase in position 4.
The substance library according to the present invention is preferably
25 distinguished by the pairing system consisting of one longer and two
shorter base strands, with the two shorter strands being complementary to
the longer strand at different points but not being complementary to one
another, and with a gap of at least one base remaining between the short
strands in the event of base-pairing with the longer strand, while at least
30 one base remains unpaired in the region of this gap corresponding to the
size thereof on the longer strand, with those bases on each of the two
shorter strands which are located at the start and at the end of the pairing
gap being linked by a linker to one molecular species in each case, while
at least one of the unpaired bases in the longer strand is linked by a linker
35 to a molecular species.
Peptides with different properties can be reversibly combined in a
controlled manner to give groups of two or three, for example by linkage to
pairing oligonucleotide ends. This means that, owing to the large number
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.
of possible combinations, orders of magnitude more different binding sites
are generated in the experiment than peptides have been synthesized.
Implementation of the principle of combinatorial variation and selection
5 under thermodynamic control, and linkage thereof, represents an
elementary technological leap in combinatorial methods: the relevant
receptor is formed by combination only in the presence of the substrate.
This receptor thus reacts to the presence of the substrate: if the latter is
10 equated with an antigen, the present system can be regarded analogously
as an "artificial immune system".
Nature has produced a remarkable number of molecules which carry out
the complex processes of the living organisms - from the immune response
15 and catalysis to signal transmission. For this it has recourse to a broad
combinatorial library of precursor molecules and checks these for the
required properties. Probably the most important example of this strategy is
the immune system which is able to generate an enormous molecular
diversity and scan the latter for receptors with high affinity and selectivity
20 for foreign antigens. The combination of molecules which intrinsically bind
only weakly, if at all, to a stable binding complex is also a principle which iswidespread in nature (heteromers [D.E. Clapham, Nature 1996, 379, 297-
299]) and whose significance for use in combinatorial chemistry has not yet
been recognized.
Fig. 1 shows diagrammatically the structure and the process of formation of
such a receptor: a short peptide chain (as library) is covalently attached via
a linker unit to the middle building block of an oligonucleotide composed,
for example, of 13 monomer building blocks. An analogous procedure is
30 applied to the two end units of the short oligonucleotides consisting of 6
monomer units.
If these three units are then offered to the substrate (ellipse), a competition
for the best binding of the peptide moieties to the substrate starts: the
35 pairing between the oligonucleotides ensures approach of the peptide
moieties in space. The reversibility of the individual steps is crucial,
resulting in exchange of the individual peptide regions until the most stable
complex has been found. This process, which takes place under
thermodynamic control, corresponds to an automatic experimental
CA 022~281~ 1998-10-28
molecular modeling. In fact, in such an experiment, all the possible
transiently occurring combinations of the three libraries is subjected to the
selection. This exchange process is temperature-dependent, i.e. exchange
of the individual strands is more frequent at higher temperature, but, at the
5 same time, the interactions of the peptide moieties with the substrate
become weaker.
After freezing of the equilibrium, covalent crosslinking of the pairing
partners, isolation and decomplexation, the receptor is obtained in free
1 0 form.
A process for the preparation of supramolecular complexes has therefore
been designed and comprises coupling compound libraries to pairing
systems.
Supramolecular complexes which have been prepared under
thermodynamic control by the processes below and selected coupled
under thermodynamic control are used when molecules or molecular
regions are to be recognized. The advantage is that the libraries, which are
20 always the same, are able very quickly in combination to solve increasingly
novel selection problems.
These are, in particular:
25 a) Molecular recognition of biologically relevant substances, i.e. diagnosis.The development of diagnostic methods in particular must keep up with the
variety of substrates to be recognized, such as metabolites or, for example,
continually mutating pathogens, so that the benefits of this process are
obvious.
b) Molecular recognition of biologically relevant substances, i.e. drug
design. The described process generates highly selective supramolecular
complexes which themselves act as active substances or as models for the
development of active substances in that, for example, they bind to, and
35 thus stimulate or block, pharmacological receptors. On the other hand, the
supramolecular complexes act as receptors in the development of active
substances, because a profile of interactions of the active substances can
be drawn up with their aid.
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c) Thermodynamically controlled constitution of catalytically active
supramolecular complexes, for example by offering transition state analogs
as substrates in the sense of the use as catalytic antibodies [L.C. Hsieh-
Wilson, X.-D. Xiang, P.G. Schultz, Acc. Chem. Res.1996, 29, 164-170].
Procedural example 1
Pyranosyl-RNA is used as pairing system (see Fig. 2), the preparation and
properties of which are well known [S. Pitsch, S. Wendeborn, B. Jaun, A.
Eschenmoser, Helv. Chim. Acta 1993, 76, 2161-2183]. Starting from D-
ribose and the nucleobases adenine and thymine, phosphoramidites
capable of coupling are prepared as described therein, and the required
hexamer and tridecamer sequences are prepared using an oligonucleotide
synthesizer. The tridecamer has the sequence 2'-MTTMT*ATATAT, one
15 hexamer has the sequence 2'-T*TMTT-4', and the other hexamer has the
'sequence 2'-ATATAT*-4', where T* is the linker nucleotide building block.
The linker nucleotide building block is synthesized by methods known from
the literature starting from the uracil nucleoside: iodination [W.-W. Sy,
Synth. Comun. 1990, 20, 3391-3394], reaction with propargyl phthalimide,
20 and hydrogenation [K.J. Gibson, S.J. Benkovic, NucleicAcids Res. 1987,
15, 6455-6467] affords the required building block. Hydrazinolysis and
iodoacetylation of the oligonucleotide takes place as described in the
literature [T. Zhu, S. Stein, Bioconjugate Chem.1994, 5, 312-315].
Tetrapeptides are prepared as compound libraries starting from
25 commercially obtainable amino acid monomers using a multiple peptide
synthesizer, providing an N-terminal cysteine residue as linker unit. The
library is divided into three portions and allowed to react in aqueous
buffered solution at room temperature in each case with the two hexamer
sequences and the tridecamer sequence to give the required conjugates,
30 which are purified by reverse phase chromatography [T. Zhu, S. Stein,
Bioconjugate Chem.1994, 5, 312-315]. Pairing of the complementary units
is detected on the basis of the decrease in the UV extinction in the pairing
experiment.
35 Procedural example 2
- Solid-phase synthesis of a CNA pentamer (Fig. 4)
The CNA oligomer was synthesized in analogy to the peptide or
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-
oligonucleotide synthesis, by stepwise incorporation of individual building
blocks on a solid phase. For this the necessary reagents were added in
excess and unreacted amounts were removed again by simple washing
steps. The polymeric support used was a polyoxyethylene (POE)/poly-
styrene copolymer (Tentagel S HMB, 0.23 mmol/g), which has good
swelling properties both in aqueous solution and in organic solvents.
The aminoethyl functionalities of the polymer were derivatized with a
hydroxymethylbenzoyl (HMB) linker; the loading with the first building block
took place using a 5-fold excess by the symmetrical anhydride method
(addition of 2.5 eq of DIC) and by adding the acylation catalyst DMAP
(2.5 eq) over the course of 20 h in DCM. The resulting loading amounted to
0.17 mmol/g. The Boc protective group of the amino functionality was
eliminated with 50% TFA in DCM, and then the resin was neutralized with
1 M DIEA/DMF. The subsequent cycles consisted of repetitive coupling of
the next monomer and elimination of the Boc protective group. The
couplings took place after preactivation of the monomer building block
(3 eq.) with the activation reagent HATU (3 eq.) in DMF (40,ul) and with
addition of 1 M DIEA/DMF (6 eq.) and 2 M lutidine/DMF (12 eq.). The
coupling times were 3-4 h at room temperature. After four coupling cycles,
the N-terminal Boc protective groups were eliminated and the pentamer
was cleaved off the resin with 2 N NaOH in methanol over the course of
15 min. The elimination solution was removed from the resin by filtration
and kept at 55~C for 2 h. Neutralization with 2 N HCI was followed by
purification with C18 RP-HPLC (Hibar prepacked column 250-4, RP-18,
5 ~um) with gradient elution (1 ml/min) from 10% to 40% B in 30 min
(solvents A: water + 0.1 % TFA, B: acetonitrile + 0.1 % TFA).
The synthesis of CNA(MTAT) was carried out with 10 mg (1.7 ,umol) of
Tentagel-HMB resin which was loaded with (S)-CNA-thymine monomer
building block. All the CNA building blocks have the S configuration. The
sequence reading from left to right corresponds to the way of writing from
the N to the C terminus usual in peptide chemistry.
CNA(MTAT): HPLC: Rt = 14.30 min; UV: ~max = 264 nm; ESI-MS:
[M + H]+Calc 1362.0, [M + 2H]2+Calc 681.0; [M + H]+eXp 1361.8, [M + 2H]2+eXp
681.5.
Desalting of the CNA pentamer CNA(MTAT) was followed by measure-
ment, in a Perkin Elmer Lambda 2 UV-VIS spectrometer, of the
temperature-dependent extinctions at 265 nm with six different concen-
trations over a range from 0 to 80~C (1.5-50 ,uM in TrisHCI buffer at pH
CA 022~281~ 1998-10-28
7.0). The first derivative of these reversible, sigmoid transition plots yields
the melting temperature (Tm = 42~C at 13,uM) (Fig. 5).
- Solid-phase synthesis of a peptide-CNA conjugate
The CNA pentamer described above was, before elimination from the
resin, extended by a dipeptide library at the N terminus. The sequence is
XO-CNA(MTAT). X represents a mixed position in which the five L-amino
acids alanine, aspartic acid, leucine, Iysine and serine are varied. O
represents a defined position, with O = L-lysine being chosen for this
sublibrary. Coupling of Boc-Lys(Fmoc)-OH to the Boc-deprotected CNA
pentamer CNA(AATAT) took place after preactivation of the amino acid
building block (6 eq.) with the activation reagent HATU (6 eq.) in DMF and
with addition of 1 M DIEA/DMF (7 eq.). The coupling time was 3 h.
Introduction of the X position took place by the split resin method. After
èlimination of the N-terminal Boc protective group,100 ,ul of DMF:DCM
(1 :1) were added to the amount of resin (5 mg), and the mixture was
divided into five portions of equal size, each of 20,ul. Coupling of the
individual amino acids took place in parallel in separate reaction vessels
with about 1 mg of oligomer-resin in each case. The individual Boc-
protected amino acids, Boc-Ala-OH, Boc-Asp(OFm)-OH, Boc-Leu-OH,
Boc-Ser-OH and Boc-Lys(Fmoc)-OH were coupled in 50-fold excess after
preactivation with HATU (50 eq.) and with addition of 1 M DIEA/DMF
(100 eq.) at room temperature for 3 h. After elimination of the N-terminal
Boc protective groups, the Fmoc protective groups were removed with 40%
piperidine/DMF (20 min). The peptide-CNA oligomer conjugates were
cleaved off the resin in each case with 2 N NaOH in methanol over the
course of 15 min. The elimination solution was removed from the resin by
filtration and kept at 55~C for 2 h. Neutralization with 2 N HCI was followed
by purification with C18-RP-HPLC (Hibar ready acid 250-4, RP-18, 5 ,um)
with gradient elution (1 ml/min) from 10% B to 40% B in 30 min (solvents A:
water + 0.1% TFA, B: acetonitrile + 0.1% TFA).
HPLC: Ala-Lys-CNA(MTAT)Rt = 15.47 min
Asp-Lys-CNA(AATAT)Rt = 15.30 min
Leu-Lys-CNA(MTAT)Rt = 16.08 min
Lys-Lys-CNA(AATAT)Rt = 15.34 min
Ser-Lys-CNA(AATAT)Rt = 15.29 min
ESI-MS: Ala-Lys-CNA(AATAT) [M + H]+Calc 1561.6; [M + H]~eXp 1561.4
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Asp-Lys-cNA(MTAT) [M + H] calc 1605.7; [M + H] exp 1605 3
Leu-Lys-CNA(MTAT) [M + H]+calc 1603.8; [M + H]+eXp 1603 4
Lys-Lys-CNA(MTAT) [M + H]+calc 1619.3; [M + H]+eXp 1619 0
Ser-Lys-CNA(MTAT) [M + H] calc 1577.7; [M + H] exp 1578 7
After characterization of the individual components, the HPLC fractions
were combined. Desalting of the peptide library CNA oligomers XLys-
CNA(MTAT) was followed by measurement, in a Perkin Elmer Lambda 2
UV-VIS spectrometer, of the temperature-dependent extinctions at 265 nm
at 50 ,uM over a range of 0-60~C (in TrisHCI buffer at pH 7.0). The first
derivative of this temperature plot yields the melting temperature (Tm =
7~C at 50 ~uM) (Fig. 6). The UV spectra of XLys-CNA(MTAT) at 0~C and
60~C differ in qualitatively the same way as the pentamer without library
CNA(MTAT). The wavelengths of the absorption maximum shifts from
261.4 nm (E = 0.3427) at 0~C to 263.8 nm (E = 0.3626) at 60~C and thus
proves the existence of the supramolecular complexes, i.e. an equilibrating
combinatorial library (Fig.7).
The meanings in this context are
Boc tert-Butyloxycarbonyl
DCM Dichloromethane
DIC Diisopropylcarbodiimide
DIEA Diisopropylethylamine
DMAP Dimethylaminopyridine
DMF Dimethylformamide
Fmoc Fluorenylmethyloxycarbonyl
HATU 0-[7-Azabenzotriazol-1-yl]-1,1,3,3-tetramethyluronium-
hexafluorophosphate
TFA Trifluoroacetic acid
