Fluorescence energy transfer substrates

Details Fluorescence energy transfer substrates Fluorescence energy transfer substrates

1995-11-17

2001-09-18

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S024000, C435S212000, C435S219000, C435S968000, C530S300000, C530S333000, C530S334000, C530S402000

Reexamination Certificate

active

06291201

ABSTRACT:

The concept of the fluorescence energy transfer (FRET) substrate for hydrolytic enzymes was first suggested by Carmel et al. (FEBS Lett., 30, 11-14(1973)); they described a trypsin substrate using naphthyl as the donor and anthracene as the acceptor. Since then, several protease substrates have been described using this principle. In this approach the donor fluorophore and acceptor molecules are positioned either side of the bond to be cleaved and are sufficiently close such that a large proportion of the fluorescence of the donor fluorophore is quenched by radiationless energy transfer to the acceptor. Cleavage causes a large increase in separation of the donor and acceptor which is made manifest by an increase in fluorescence. Here the acceptor moiety does not need to be a fluorophore; the key requirement is that the absorption maximum should be the same as, or close to, the emission maximum of the donor fluorophore. Optionally, the acceptor may be itself a fluorophore, in which case the enzymic reaction may be followed by the decrease in energy transfer fluorescence of the acceptor fluorophore. A variety of donors and acceptors have been employed, a common pair is an “in-sequence” trytophan as the donor and dansyl as the acceptor. Energy transfer substrates are particularly useful for proteases that have significant recognition in the native substrate of amino acids C-terminal to the cleavage site; other proteases that do not have such recognition sites are conveniently measured using peptides with fluorogenic or chromogenic leaving groups.
Present FRET substrates are limited to fluorophores that require UV excitation. This limits their use especially in compound screening methods. Furthermore the donor and/or acceptor species are attached during synthesis of the peptide substrate. This generally requires specialist reagents and know-how relating to their use. Accordingly a range of donor and acceptor species cannot be readily assessed for a given substrate.
Wang et al (Tet. Lett., 1990, 13, 6493-6496) describe a method for the preparation of fluorogenic HIV protease substrates which involves the addition of donor and acceptor species to the N- and C-termini of a peptide substrate. However, as the authors acknowledge, the procedures followed which include fluorophore attachment to the C-terminus are not straightforward. Overall this paper does not disclose a generally applicable approach.
Maggiora et al (J. Med. Chem. 1992, 35, 3727-3730) describe a method for the synthesis of FRET substrates which employ a (donor) EDANS labelled glutamic acid derivative which is introduced during peptide synthesis. The disadvantage of this approach is that, if for example for reasons of poor activity, the EDANS derivative does not give a useful substrate, then a different amino acid derivative needs to be prepared and the peptide synthesised afresh. Selecting the optimum donor is therefore not straightforward.
We have now devised a novel and advantageous method for preparing FRET protease substrates wherein, after synthesis of the polypeptide substrate, a donor or acceptor is attached via the side chain of an amino acid comprised in the polypeptide. This allows a wide range of donor and acceptor pairs, many of which are commercially available, to be attached to the peptide structure. Accordingly, desirable donor and acceptor combinations may be readily selected.
In a first aspect of the present invention we provide a method for the preparation of a polypeptide substrate for proteolytic cleavage and having a donor or acceptor species attached via a side chain of an amino acid therein which method comprises contacting an appropriate reactive donor or acceptor species with a polypeptide substrate having a side chain of an amino acid therein adapted for reaction therewith.
The above method is preferably used to prepare FRET protease substrates having appropriate donor and acceptor species on opposite sides of a proteolytic cleavage site. This is achieved either by prior or subsequent reaction of the polypeptide substrate with the corresponding reactive donor or acceptor species. By “corresponding” we mean that if a donor is attached first, then a suitable energy transfer acceptor is subsequently attached or vice versa. If desired both donor and acceptor species may be attached via the side chains of amino acids therein.
The polypeptide substrate is conveniently prepared by direct synthesis, for example as outlined in Solid Phase Peptide Synthesis, E Atherton and R C Sheppard, IRL Press, 1989. The polypeptide substrate may comprise natural or non-natural (for example D-) amino acids joined by natural or modified peptide linkages, or any combination thereof.
The sites on the polypeptide substrate for attachment of reactive donor and acceptor species are conveniently provided by amino and thiol groups. More conveniently these groups are comprised in the N-terminus and the side chain of a cysteine residue respectively. Where no appropriate group is naturally present in the sequence, this may be introduced at any convenient point, preferably by replacement of a non-critical residue. By way of example the thiol group may be introduced by replacement of a non-critical residue by cysteine. It will be clear that by “adapted for reaction” we include amino acids which are either inherently adapted for reaction or chemically modified.
Alternatively, the sites on the polypeptide substrate may be provided by introduction of chemically modified residues during peptide synthesis. By “attachment via the side chain of an amino acid” we therefore include attachment via a side chain of any amino acid, whether modified or replaced by any convenient chemical linkage. We do not exclude attachment via any convenient linkage replacing the hydrogen atom at the &agr;-carbon of the amino acid residue.
It will be appreciated that the donor and acceptor species and their means of attachment are selected such that proteolytic cleavage of the substrate is not affected to any significant extent. In general, the N-terminus of the selected peptide is chosen to be a sufficient distance from the site of proteolytic cleavage such that its modification does not significantly affect activity. This will vary from enzyme to enzyme, however, by way of example, the N terminus is conveniently at least 3 amino acids from the cleavage site, more conveniently more than 5. Likewise the point of attachment for the donor/acceptor on the side chain is selected so as not to significantly affect activity. This will also vary according to the enzyme, but by way of example, it should be at least 3, preferably 5 amino acids away from the site for proteolytic cleavage. The total distance between attachment sites needs to be such that a useful amount of energy transfer can occur. Whilst we do not wish to be bound by theoretical considerations, this will depend on the 3 dimensional conformation adopted by the peptide in solution. The worst case can be predicted by assuming both a linear structure, and that each amino acid accounts for 3.8 angstroms. Hence for 50% energy transfer and assuming a favourable alignment of dipoles, the number of amino acids separating the donor and acceptor should be less than Ro/3.8, where Ro is the distance giving 50% transfer. For a fuller discussion, see for example J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, 1983. Appropriate experiments for determining the optimum positions for attachment of the donor and acceptor species will be apparent to the scientist of ordinary skill.
Where amino and thiol chemistry is employed, the polypetide substrate is conveniently firstly contacted with a donor or acceptor species so as to attach this via the side chain of an amino acid therein. The resulting substrate is then contacted with a corresponding donor or acceptor species so as to provide a FRET protease substrate.
Therefore in a preferred aspect of the present invention we provide a method for the preparation of a FRET protease substrate having donor and acceptor species on opposite sides of a proteolytic cl

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