Reagent and method for determining activity of herpes protease

A class of compounds is described for use as fluorogenic substrates for herpesvirus proteases and for use in an assay to identify inhibitors of herpesvirus proteases. Compounds of particular interest are defined by Formula II ##STR1## wherein W is selected from glycine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid and 7-aminoheptanoic acid; wherein Y is selected from EDANS, Abz, DANSYL, nicotinic acid, 4-guanidino-benzoic acid, N-methyl-Abz, 4-chloro-Abz, 5-chloro-Abz, 6-chloro-Abz, 3,5-dibromo-Abz, 6-amino-nicotinic acid, 2-aminonicotinic acid, 2-chloronicotinic acid, niflumic acid and fluorogenic derivatives thereof; and wherein Z is selected from tryptophan, tyrosine, phenylalanine, p-nitrophenylalanine, m-nitrophenylalanine, DABSYL, DABCYL and halogenated derivatives thereof.

FIELD OF THE INVENTION 
This invention relates to reagents and a method for determining the 
activity of herpesvirus proteases. More particularly, the invention 
relates to fluorogenic substrates and a fluorometric assay for CMV 
protease and other herpesvirus proteases. 
BACKGROUND OF THE INVENTION 
There is a great need for new therapies active in the treatment of viral 
diseases. Whereas there has been great progress in developing a variety of 
therapies for the treatment of bacterial infections, there are few viable 
therapies for the treatment of viruses in general and herpesvirus in 
particular. Herpesviridae is a family of DNA viruses which include herpes 
simplex virus type-1 (HSV-1) herpes simplex virus type-2 (HSV-2), 
cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus, 
human herpesvirus-6 (HHV6), human herpesvirus-7 (HHV7), pseudorabies and 
rhinotracheitis, among others. Ganciclovir, acyclovir and foscarnet are 
currently utilized for the treatment of herpesvirus infections. However, 
these therapies can have substantial side effects based on their 
deleterious effects on host cell DNA replication, or they can be used to 
treat only a limited number of herpesviruses. In addition, herpesviruses 
are known to develop resistance to these therapies which causes a 
progressive decline in efficacy [Ljungman et al., J. Infect. Dis., 162, 
244 (1990) and Gately et al, J. Infect. Dis., 161, 711 (1990)]. 
It is known that herpesviruses express their genetic content by directing 
the synthesis of a number of proteins encoded by the herpesvirus DNA in 
the host cell. One of the important virus encoded proteins is made as a 
precursor consisting of an amino terminal-located protease and carboxyl 
terminal-located assembly protein. This precursor is proteolytically 
processed in an autocatalytic manner at a specific amino acid sequence 
known as the "release" site yielding separate protease and assembly 
protein. The assembly protein is cleaved further by the protease at 
another specific amino acid sequence known as the "maturation" cleavage 
site. Recently, EP No. 514,830, published Nov. 25, 1992, describes a 
virus-specific serine protease which has a role in herpesvirus 
replication. Additionally, Lui and Roizman (J. Virol, 65, 5149 (1991)) 
describe the sequence and activity of a protease and the associated 
assembly protein encoded by U.sub.L 26 of HSV-1. A. R. Welch et al (Proc. 
Natl. Acad. Sci. USA, 88, 10792 (1991)) describe the related protease 
(also known as assemblin) and assembly protein encoded by U.sub.L 80 of 
CMV. An approach currently being investigated for potential use in the 
treatment of herpesvirus infections is the development of inhibitors of 
herpesvirus proteases. 
Various inhibitors of herpesvirus proteases are described in applicants' 
copending application. 
In order to facilitate the rapid identification of herpesvirus protease 
inhibitors, an assay which allows for high throughput and linearity is 
desirable. Initial assays used in the characterization of herpesvirus 
proteases have been based on electrophoretic separation of products. See 
EP 514,830. Such method is impractical for screening large numbers of 
enzymatic inhibitors. An assay which allows for quantitative kinetic 
characterization of the interaction of the inhibitors with herpesvirus 
proteases is more preferred. 
Resonance energy transfer has been utilized in the biochemical study of 
hydrolyric enzymes because of the technique's high sensitivity. See e.g. 
Guilbault, Enzymatic Methods of Analysis, 43-47 (1970). 
G. Marshall and M. Toth, in U.S. Pat. No. 5,011,910, describe the use of 
fluorogenic substrates for the determination of HIV proteases. S. 
Netzell-Arnett et al (Anal. Biochem., 195, 86 (1991)) describe the use of 
a fluorescent assay for human matrix metalloproteinases. 
C. Garcia-Echeverria and D. Rich describe the use of quenched fluorogenic 
peptide substrates in an investigation of the sensitivity of the cysteine 
protease, papain, to modifications in the P1' and P2' sites (FEBS Lett., 
297, 100 (1992), and Biochem. and Biophys. Res. Comm., 187, 615 (1992)). 
E. Matayoshi et al describe the use of an EDANS/DABCYL-containing 
substrate for assaying HIV protease (Science, 247, 954 (1990)). G. Wang et 
al use similar groups on their related substrate (Tet. Lett., 31, 6493 
(1990)). L. Maggiora et al describe a solid-phase peptide synthesis method 
of preparing EDANS/DABCYL-containing substrates (J. Med. Chem., 35, 3727 
(1992)).

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, fluorogenic substrates and a 
fluorometric assay for herpesvirus protease, and preferably the CMV 
protease (also known as assemblin) encoded by U.sub.L 80, utilizing these 
substrates are provided. 
A class of compounds useful as substrates for herpesvirus proteases is 
defined by Formula I 
EQU Z-(W).sub.m -X-(V).sub.n -.sup.Y (I) 
wherein X is an amino acid sequence sufficient for substrate recognition by 
a herpesvirus protease; wherein V and W are independently selected from 
aminoalkylcarboxylic acids; wherein m and n are numbers independently 
selected from 0 and 1; and wherein one of Y and Z is a fluorogenic donor 
radical and the other is an acceptor radical. 
A preferred class of compounds consists of those compounds of Formula I 
wherein X is a peptide containing six to sixteen amino acids, inclusive; 
wherein V and W are independently selected fromlower aminoalkylcarboxylic 
acids; wherein the fluorogenic donor radical is a fluorescence emitting 
radical; and wherein the acceptor radical is an aromatic radical which 
absorbs the fluorescence energy of the fluorogenic donor radical when the 
acceptor radical and the fluorogenic donor radical are covalently held in 
close proximity. 
A more preferred class of compounds consists of those compounds of Formula 
I, wherein X is selected from -Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID 
NO:1], -Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID NO:2], 
-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ ID NO:3], 
-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID NO:4], 
-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ ID NO:5], 
-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr- [SEQ ID 
NO:6],-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID NO:7], 
-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ ID NO:8], 
-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr- [SEQ ID NO:9], 
-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala- [SEQ ID NO:10], 
-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID NO:11], 
-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ ID NO:12], 
-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr- [SEQ ID NO:13], 
-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala- [SEQ ID NO:14], 
-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID NO:15], 
-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala [SEQ ID NO:16], 
-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr- [SEQ ID NO:17], 
-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala- [SEQ ID NO:18], 
-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID NO:19], 
-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ ID NO:20], 
-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr- [SEQ ID NO:21], 
-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala- [SEQ ID NO:22], 
-Ala-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID 
NO:23], -Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ 
ID NO:24], -Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr- 
[SEQ ID NO:25], 
-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala- [SEQ ID 
NO:26], -Val-Ala-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- 
[SEQ ID NO:27], 
-Ala-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ ID 
NO:28], -Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr- 
[SEQ ID NO:29]and 
-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-Thr-Ala- [SEQ ID 
NO:30]and homologs thereof; wherein V and W are independently selected 
from glycine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic 
acid and 7-aminoheptanoic acid; wherein the fluorogenic radical is 
selected from EDANS, 2-aminobenzoic acid (Abz), 
5-dimethylamino-naphthalene-1-sulfonyl (DANSYL), nicotinic acid, 
4-guanidino-benzoic acid, N-methyl-Abz, 4-chloro-Abz, 5-chloro-Abz, 
6-chloro-Abz, 3,5-dibromo-Abz, 6-amino-nicotinic acid, 2-amino-nicotinic 
acid, 2-chloronicotinic acid, niflumic acid and fluorogenic derivatives 
thereof; and wherein the acceptor radical is selected from tryptophan, 
tyrosine, phenylalanine, p-nitrophenylalanine, m-nitrophenylalanine, 
DABSYL, DABCYL and halogenated derivatives thereof. 
Within Formula I there is a subclass of compounds of high interest 
represented by Formula II 
______________________________________ 
Z--W--Gly--Val--Val--Asn-- 
Ala--Ser--Ala--Arg--Leu--Ala--Y (II) 
[SEQ ID NO:31] 
______________________________________ 
wherein W is an aminoalkylcarboxylic acid; wherein one of Y and Z is a 
fluorogenic radical and the other is an acceptor radical. 
A preferred class of compounds consists of those compounds of Formula II 
wherein W is a lower aminoalkylcarboxylic acid; wherein Y is a fluorogenic 
radical; and wherein Z is an acceptor radical. 
A more preferred class of compounds consists of those compounds.of Formula 
II wherein W is selected from glycine, 4-aminobutyric acid, 
5-aminopentanoic acid, 6-aminocaproic acid and 7-aminoheptanoic acid; 
wherein Y is selected from EDANS, Abz, DANSYL, nicotinic acid, 
4-guanidino-benzoic acid, N-methyl-Abz, 4-chloro-Abz, 5-chloro-Abz, 
6-chloro-Abz, 3,5-dibromo-Abz, 6-amino-nicotinic acid, 2-amino-nicotinic 
acid, 2-chloronicotinic acid, niflumic acid and fluorogenic derivatives 
thereof; and wherein Z is selected from tryptophan, tyrosine, 
phenylalanine, p-nitrophenylalanine, m-nitrophenylalanine, DABSYL, DABCYL 
and halogenated derivatives thereof. 
A compound of Formula II of particular interest is 
DABCYL-.gamma.-Abu-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-EDANS 
(designated DE2) [SEQ ID NO:32]. 
Within Formula I there is a second subclass of compounds of high interest 
represented by Formula III 
##STR2## 
wherein W is an aminoalkylcarboxylic acid; wherein one of Y and Z is a 
fluorogenic radical and the other is an acceptor radical. 
A preferred class of compounds consists of those compounds of Formula III 
wherein W is a lower aminoalkylcarboxylic acid; wherein Y is a fluorogenic 
radical; and wherein Z is an acceptor radical. 
A more preferred class of compounds consists of those compounds of Formula 
III wherein W is selected from glycine, 4-aminobutyric acid, 
5-aminopentanoic acid, 6-aminocaproic acid and 7-aminoheptanoic acid; 
wherein Y is selected from EDANS, Abz, DANSYL, nicotinic acid, 
4-guanidino-benzoic acid, N-methyl-Abz, 4-chloro-Abz, 5-chloro-Abz, 
6-chloro-Abz, 3,5-dibromo-Abz, 6-amino-nicotinic acid, 2-amino-nicotinic 
acid, 2-chloronicotinic acid, niflumic acid and fluorogenic derivatives 
thereof; and wherein Z is selected from tryptophan, tyrosine, 
phenylalanine, p-nitrophenylalanine, m-nitrophenylalanine, DABSYL, DABCYL 
and halogenated derivatives thereof. 
A compound of Formula III of particular interest is 
DABCYL-.gamma.-Abu-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-EDANS (designated DE1) 
[SEQ ID NO:34]. 
An amino acid sequence sufficient for substrate recognition by a 
herpesvirus protease includes "maturation" cleavage site sequences and 
"release" cleavage site sequences of herpesvirus protease substrates. 
These include "maturation" and "release" cleavage site sequences for HCMV, 
HSV-1, HSV-2, VZV, HHV-6, HHV-7 and EBV proteases. The preferred novel 
substrates are based on a HCMV "maturation" cleavage site sequence 
(Val-Ala-Glu-Arg-Ala-Gln-Ala-Gly-Val-Val-Asn-Ala*Ser-CYs-Arg-Leu-Ala-Thr-A 
la [SEQ ID NO:35], where "*" denotes the cleavage site) at the C-terminus 
of the capsid assembly protein. Peptides of various lengths encompassing 
this sequence, or homologs thereof, provide amino acid sequences 
sufficient for substrate recognition by a herpesvirus protease. Preferably 
peptides have between six amino acids and twelve amino acids. This length 
also provides an adequate distance for the acceptor radical to 
intramolecularly quench the fluorogenic radical. Two sequences, an 
octapeptide -Val-Val-Asn-Ala-Ser-Ala-Arg-Leu- [SEQ ID NO:l]and a 
decapeptide -Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala- [SEQ ID NO:5]are 
even more preferred. 
The fluorometric assay developed to incorporate the substrate compounds of 
the invention is a method for the determination of a herpesvirus protease 
and inhibitors of a herpesvirus protease, e.g. a CMV protease (also known 
as assemblin) encoded by U.sub.L 80, which comprises placing a known 
amount of the herpesvirus protease in the wells of a "Microlite 1"96 well 
plate (Dynatek Laboratories, Inc.), adding a known amount of the substrate 
at 22.degree. C., measuring the change in fluorescence with time and 
determining the molar velocity of the herpesvirus protease by comparing 
against a standard curve which relates changes in fluorescent intensity to 
changes in concentration of product. Potential inhibitors of the protease 
action can be added to the well to observe their effect on the protease 
cleavage activity. 
Besides variations in the fluorescence donor and acceptor radicals, the 
amino acids in the substrate sequences can be varied to optimize the 
affinity and kinetic properties for the particular herpesvirus protease 
under consideration. In the case of assemblin, valine (P3 - Val) can be 
replaced by Leu, Phe and other hydrophobic residues. Leucine (P4'- Leu) 
can similarly be substituted with Val and other hydrophobic residues. 
Cysteine (P2'- Cys) can be replaced with Ala. 
The acceptor radical can be located at other positions in the N-terminal 
section of the sequence such as by replacing Val (P3) with a modified 
amino acid radical such as Phe(m-NO.sub.2). The C-terminal acid can be 
replaced by an acceptor radical such as nitrobenzylamine as well. This may 
be necessary, in order to accommodate particular substrate requirements of 
a given herpesvirus protease. Attachment of aminobenzoic acid and 
substituted aminobenzoic acid radicals is preferred at the N-terminus, 
however, addition of a lysine amino acid at the C-terminus will allow 
their incorporation at the C-terminus portion of the substrate. 
Where the term "fluorogenic donor" is used, it embraces a fluorescence 
emitting radical, such as anthracene, aminobenzoyl, indole, and 
aminoethylnaphthyl radicals, and the like, which can be modified and 
attached to the amino acid sequence. Such radicals include 
5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid (EDANS), 
2-aminobenzoic acid (Abz) and derivatives thereof, e.g. N-methyl-Abz, 
4-chloro-Abz, 5-chloro-Abz, 6-chloro-Abz, 3,5-dibromo-Abz, 
5-dimethylamino-naphthalene-1-sulfonyl (DANSYL) and derivatives thereof, 
nicotinic acid and derivatives thereof, such as 6-aminonicotinic acid, 
2-aminonicotinic acid, 2-chloronicotinic acid, and niflumic acid, 
4-guanidino-benzoic acid and derivatives of 4-guanidino-benzoic acid; and 
the like. The term "acceptor radical" embraces radicals which have a 
quenching aromatic radical which absorbs the fluorescence energy of the 
fluorogenic donor radical and reduces the fluorescence emission when the 
fluorogenic donor radical is covalently held in close proximity to the 
acceptor radical. Such "acceptor radicals" include 
4-(4'-dimethylaminophenylazo)benzoyl (DABCYL), 
4-(4'-dimethylaminophenylazo) phenylsulfonyl (DABSYL), dinitrophenol 
(DNP), or an amino acid derivative such as tryptophan (Trp), tyrosine 
(Tyr), phenylalanine (Phe), p-nitrophenylalanine [Phe(p-NO.sub.2)], 
nitrotyrosine, m-nitrophenylalanine [Phe(m-NO.sub.2)], and halogenated 
derivatives thereof. The term "aminoalkylcarboxylic acids" embraces 
radicals which can be included between either the fluorescing donor 
radical or the acceptor radical, and the peptide sequences. Such radicals 
act as spacers and reduce the possibility of the fluorescing donor radical 
or the acceptor radical, having a steric or other negative effect on the 
binding of the substrate and the enzyme. Such aminoalkylcarboxylic acids 
embrace linear or branched radicals having one to about twenty carbon 
atoms or, preferably, one to about twelve carbon atoms. More preferred 
aminoalkylcarboxylic acids radicals are "lower aminoalkylcarboxylic acids" 
radicals having one to about ten carbon atoms. Most preferred are lower 
aminoalkylcarboxylic acids radicals having one to about seven carbon 
atoms. Examples of such radicals include glycine, 4-aminobutyric acid, 
5-aminopentanoic acid, 6-aminocaproic acid, 7-aminoheptanoic acid, and the 
like. 
It will be appreciated that various modifications can be made to the 
aforesaid preferred fluorogenic substrates to provide substantially 
similar useful results in the fluorometric assay for herpesvirus protease. 
While the specification concludes with claims particularly pointing out and 
distinctly claiming the subject matter regarded as forming the present 
invention, it is believed that the invention will be better understood 
from the following detailed description of preferred embodiments of the 
invention taken in conjunction with the appended drawings, in which, 
briefly: 
FIG. 1 is a graphical representation which shows the fluorescence emission 
spectra of 2 .mu.M substrate DE1 [SEQ ID NO:34]and 2 .mu.M 
Ser-Ala-Arg-Leu-EDANS (the C-terminal cleavage product) [SEQ ID NO:36]. 
Fluorescence magnitude is plotted versus wavelength in nm. 
FIG. 2 is a graphical representation which shows the time course of change 
in fluorescence emission spectra during hydrolysis of substrate DE1 [SEQ 
ID NO:34]by CMV assemblin. The arrow indicates the addition of protease to 
10 .mu.M DE1 [SEQ ID NO:34]. The cleavage of the substrate resulted in a 
10-fold increase in fluorescence emission at 340 nm excitation and 480 nm 
emission wavelength within 30 minutes. Fluorescence magnitude is plotted 
versus time. 
GENERAL SYNTHETIC PROCEDURES 
The preferred novel fluorogenic substrates of this invention and their 
analogs can be made by known solution and solid phase peptide synthesis 
methods but modified to incorporate the acceptor radical, e.g. DABCYL at 
the N-terminal position, the fluorogenic radical, e.g. EDANS at the C 
terminus and spacer radicals between the peptide and the acceptor radical 
or the fluorogenic radical. The preferred peptide synthesis method follows 
conventional Merrifield solid-phase procedure [J.Amer. Chem. Soc., 85, 
2149 (1963); Science, 150, 178 (1965) modified by the procedure of Tam et 
al., J. Amer. Chem. Soc., 105, 6442 (1983)]. 
In order to illustrate specific preferred embodiments of the invention in 
greater detail, the following exemplary laboratory preparative work was 
carried out. It should be understood that the invention is not limited to 
these specific examples. 
Solid phase synthesis of assemblin substrates are prepared by conventional 
solid phase peptide synthesis using an appropriate Boc-amino acid 
phenylacetamidomethyl (PAM) resin. For each synthesis, 0.5 grams of resin 
was used (0.5 mmole). The following synthetic protocol is an example of 
that can be used for incorporation of the Bocamino acids. 
______________________________________ 
Deprotection: 
50% trifluoroacetic acid/CH.sub.2 Cl.sub.2 5 minutes/25 minutes 
CH.sub.2 CL.sub.2 
2 .times. 1 minutes 
Isopropanol 2 .times. 1 minutes 
CH.sub.2 Cl.sub.2 
2 .times. 1 minutes 
Neutralization: 
10% diisopropylethylamine/CH.sub.2 Cl.sub.2 3 minutes/5 minutes 
CH.sub.2 Cl.sub.2 
2 .times. 1 minutes 
DMF 2 .times. 1 minutes 
______________________________________ 
Amino acids are coupled to the resin, or the growing peptide chain on the 
resin, by adding 4-equivalents of butyloxycarbonyl (Boc) protected amino 
acid and 4 equivalents of diisopropylcarbodiimide (DCC) in the presence of 
4 equivalents of hydroxybenzotriazole (HOBT) in dimethylformamide (DMF) 
for 2 hours. 4-(4'-Dimethylaminophenylazo)benzoyl (DABCYL) is obtained 
from Sigma as the free acid and coupled in the regular fashion. Completed 
peptides are cleaved by the hydrofluoric acid (HF)/anisole 9:1 procedure 
of Tam et al., J.Amer. Chem. Soc., 105, 6442 (1983). Crude DABCYL-peptides 
are dissolved in 20% acetic acid and lyophilized. Crude peptides are 
purified by reverse-phase HPLC on a C.sub.18 semipreparative column using 
a 0.1% trifluoroacetic acid (TFA) and acetonitrile gradient. The EDANS 
radical was coupled to DABCYL-peptide free acid in DMF by adding a 5 times 
molar excess of EDANS in the presence of 
benzotriazolyl-N-oxytris(dimethylamino)-phosphonium hexafluorophosphate 
(BOP) and diisopropylethylamine (DIPEA). The mixture was filtered and 
diluted with 5 mL 50% acetic acid and 20 mL water. Crude 
DABCYL/EDANS-peptides are purified by reverse-phase HPLC on a C.sub.18 
semipreparative column using a 0.1% TFA and acetonitrile gradient. Their 
identity was confirmed by high-resolution mass spectrometry and amino acid 
analysis. 
EXAMPLE 1 
##STR3## 
DABCYL-.gamma.-Abu-Val-Val-Asn-Ala-Ser-Ala-Arq-Leu-EDANS (DE1) [SEQ ID 
NO:34] 
DABCYL-.gamma.-Abu-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-EDANS (DE1) [SEQ ID 
NO:34]was synthesized on an Applied Biosystems peptide synthesizer (Model 
430A) using a standard synthesis protocol, starting with 0.5 mmol 
Boc-Leu-PAM resin. The fully protected DABCYL-.gamma.-Abu-peptide resin 
was cleaved and deprotected with treatment of HF/anisole (9:1) at 
0.degree. C. for 1 hour. Crude DABCYL-.gamma.-Abu-peptide was purified on 
HPLC using acetonitrile/water (0.1% TFA) gradient (20-50% acetonitrile in 
30 minutes). Purified DABCYL-.gamma.-Abu-peptide free acid (24 mg, 20 
.mu.mol) was dissolved in 5 mL DMF and coupled with 28.8 mg (100 .mu.mol) 
EDANS in the presence of 44 mg (100 .mu.mol) BOP and 35 .mu.L (200 
.mu.mol) DIPEA for 12 hours. The reaction mixture was filtered and diluted 
with 5 mL 50% acetic acid and 20 mL water. This solution was purified on 
HPLC using acetonitrile/water (0.1% TFA) gradient (20-50% acetonitrile in 
30 minutes). The identity of 
DABCYL-.gamma.-Abu-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-EDANS (DE1) [SEQ ID 
NO:34]was confirmed by amino acid analysis and FAB mass spectrometry. FAB 
MS: (M+H)+=1413.8 
EXAMPLE 2 
##STR4## 
DABCYL-.gamma.-Abu-Gly-Val-Val-Asn-Ala-Ser-Ala-Arq-Leu-Ala-EDANS (DE2) 
[SEQ ID NO: 32 ] 
DABCYL-.gamma.-Abu-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-EDANS (DE2) [SEQ 
ID NO:32]was synthesized on an Applied Biosystems peptide synthesizer 
(Model 430A) using a standard synthesis protocol, starting with 0.5 mmol 
Boc-Ala-PAM resin. The fully protected DABCYL-.gamma.-Abu-peptide resin 
was cleaved and deprotected with treatment of HF/anisole (9:1) at 
0.degree. C. for 1 hour. Crude DABCYL-.gamma.-Abu-peptide was purified on 
HPLC using acetonitrile/water (0o1% TFA) gradient (20-50% acetonitrile in 
30 minutes). Purified DABCYL-.gamma.-Abu-peptide free acid in 5 mL DMF (26 
mg, 20 .mu.mol) was coupled with 28.8 mg (100 .mu.mol) EDANS in the 
presence of 44 mg (100 .mu.mol) BOP and 35 .mu.L (200 .mu.mol) DIPEA for 
12 hour. The reaction mixture was filtered and diluted with 5 mL 50% 
acetic acid and 20 mL water. This solution was purified on HPLC using 
acetonitrile/water (0.1% TFA) gradient (20-50% acetonitrile in 30 
minutes). The identity of 
DABCYL-.gamma.-Abu-Gly-Val-Val-Asn-Ala-Ser-Ala-Arg-Leu-Ala-EDANS (DE2) 
[SEQ ID NO:32]was confirmed by amino acid analysis and FAB mass 
spectrometry. FAB MS: (M+H)+=1541.8 
The emission spectra of DE1 [SEQ ID NO:34]and its C-terminal cleavage 
product, Ser-Ala-Arg-Leu-EDANS [SEQ ID NO:38], were measured on a 
Perkin-Elmer LS50 spectrophotometer. The substrates of the invention 
showed a small amount of fluorescent background due to incomplete 
quenching of the fluorogenic donor radical. As shown in FIG. 1, the 
C-terminal cleavage product had a broad emssion maximum between 470 nm and 
490 nm and the fluorescence magnitude was 20-fold greater that of an equal 
concentration of DE1 [SEQ ID NO:34]. 
An assay was developed for screening potential herpesvirus protease 
inhibitors utilizing the substrates of the invention. Recombinant HCMV 
protease was purified from E. coli expressing a DNA construction encoding 
the protease domain of the U.sub.L 80 open reading frame of human 
cytomegalovirus strain AD169. The construction also encoded six additional 
histidine residues at the amino terminus of the protease. These additional 
histidine residues provided an affinity ligand by which it was purified 
using nickel-nitriloacetic acidagarose (Qiagen). 
The purified protease was stored as a 1-3 mg/ml stock solution in 50 mM 
sodium phosphate buffer, pH 7.4; 300 mM sodium chloride; 100 mM imidazole; 
50% (v/v) glycerol. This stock was diluted with assay buffer to 4.8 
.mu.g/ml. A 100 .mu.L aliquot of this solution was used in the enzyme 
reaction. 
A specific substrate was synthesized based on the cleavage specificity of 
HCMV protease at the "maturation" site of the assembly protein. Upon 
excitation at 360 nm the EDANS chromophore emitted light (fluoresces) at 
490 nm that was absorbed by the DABCYL chromophore (E.sub.max =460 nm). 
However, when the two chromophores were separated because of hydrolysis of 
the peptide moiety by HCMV protease, the EDANS fluorescence was no longer 
quenched and an increase in fluorescence was realized. Substrate was 
stored as a stock solution at 160 .mu.g/ml in dimethyl sulfoxide. This was 
diluted 10-fold with assay buffer to give a concentration of 16 .mu.g/ml 
just before use. An aliquot of 50 .mu.L was used in the reaction 
An assay Buffer (10 mM sodium phosphate buffer, pH 7.4; 150 mM sodium 
acetate; 0.1% CHAPS; and 20% (v/v) glycerol) was used to dilute stock 
solutions of enzyme and substrate. 
Inhibitors were dissolved in dimethyl sulfoxide at 16-times the final assay 
concentration. A 10 .mu.L aliquot of this inhibitor solution was used in 
the reaction. 
A 100 .mu.L aliquot of enzyme solution (4.8 g/ml) was mixed with 10 .mu.L 
of inhibitor solution in a 96-well plate and incubated at 22.degree. C. 
for 30 minutes. Control reactions contained 10 .mu.L of dimethylsulfoxide 
instead of inhibitor solution. After the preincubation was completed, 50 
.mu.L of substrate solution was added and the fluorescence recorded every 
5 minutes for a period of 30 minutes. All reactions were run in triplicate 
with the exception of the uninhibited control reaction which was 
replicated 6-fold. The change in fluorescence was recorded over time and 
an average rate for each set of 3 reactions was calculated. Fluorescence 
values were subject to spurious variations due to factors such as dust in 
the 96well plate; such results were omitted from the calculation of 
relative rates. 
Although this invention has been described with respect to specific 
embodiments, the details of these embodiments are not to be construed as 
limitations. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 36 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /note="Xaa at position 2 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 8 is any hydrophobic amino 
acid. Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 8. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and Xaa at position 8." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ValXaaAsnAlaSerXaaArgXa a 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note="Xaa at position 3 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 6 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 8 is any hydrophobic amino 
acid. Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 8. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and Xaa at position 8." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GlyValXaaAsnAlaSerXaaArgXaa 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /note="Xaa at position 2 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 9. V=an 
aminocarboxylic acid which may or may not be 
inserted between Y and amino acid at position 9." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ValXaaAsnAlaSerXaaArgXaaA la 
15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalklylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /note="Xaa at position 4 is any 
hydrophic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 10 is any hydrophobic 
amino acid. Y=a fluorogenic donor or acceptor 
radical which follows Xaa at position 10. V= 
an aminoalkylcarboxylic acid which may or may not 
be inserted between Y and Xaa at position 10." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AlaGlyValXaaAsnAlaSerXaaArgXaa 
15 10 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note="Xaa at position 3 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 10. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 10." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GlyVal XaaAsnAlaSerXaaArgXaaAla 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /note="Xaa at position 2 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is any 
hydrophobic residue." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 10. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 10." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ValXaaAsnAlaSerXaaArgXaaAlaThr 
1510 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
( B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 5 
(D) OTHER INFORMATION: /note="Xaa at position 5 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 11 is any hydrophobic 
amino acid. Y=a fluorogenic donor or acceptor 
radical which follows Xaa at position 11. V= 
an aminoalkylcarboxylic acid which may or 
may not be inserted between Y and Xaa at 
position 11." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GlnAlaGlyValXaaAsnAlaSerXaaArgXaa 
1510 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /note="Xaa at position 4 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 11. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 11." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AlaGlyValXaaAsnAlaSerXaaArgXaaAla 
1 510 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note="Xaa at position 3 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 11. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
11." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GlyValXaaAsnAlaSerXaaArgXaaAlaThr 
1510 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /note="Xaa at position 2 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is Ala or 
Cys." 
( ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 11. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
11." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ValXaaAsnAlaSerXaaArgXaaAlaThrAla 
15 10 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 12 is any hydrophobic 
amino acid. Y=a fluorogenic donor or acceptor 
radical which follows the amino acid at position 
12. V=an aminoalkylcarboxylic acid which may or 
may not be inserted between Y and Xaa at position 
12." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
AlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaa 
1 510 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 5 
(D) OTHER INFORMATION: /note="Xaa at position 5 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note="Xaa at position 11 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic acceptor or donor radical 
which follows the amino acid at position 12. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
12." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GlnAlaGlyValXaaAsnAlaSerXaaArgXaaAla 
1510 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /note="Xaa at position 4 is 
any hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 12. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
12." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AlaGlyValXaaAsnAlaSerXaaArgXaaAlaThr 
15 10 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/ note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note="Xaa at position 3 is any 
hydrophobic amino acid." 
(ix ) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 12. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
12." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GlyValXaaAsnAlaSerXaaArgXaaAlaThrAla 
1510 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note="Xaa at position 11 is Ala 
or Cys." 
(ix) FEATURE: 
( A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 13 is any hydrophobic 
amino acid. Y=a fluorogenic donor or acceptor 
radical which follows Xaa at position 13. V= 
an aminoalkylcarboxylic acid which may or may 
not be inserted between Y and Xaa at position 
13." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
ArgAlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaa 
1510 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D ) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /note="Xaa at position 12 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 13. V=an 
aminoalkylcarboxylic acid which may or may be 
inserted between Y and the amino acid at position 
13." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
AlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaaAla 
15 10 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note=" Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 5 
(D) OTHER INFORMATION: /note="Xaa at position 5 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note="Xaa at position 11 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 13. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
13." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GlnAlaGlyValXaaAsnAlaSerXaaArgXaaAlaThr 
1510 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
( D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /note="Xaa at position 4 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y= a fluorogenic donor or acceptor radical 
which follows the amino acid at position 18. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
18." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
AlaGlyValXaaAsnAlaSerXaaArgXaaAlaThrAla 
1 510 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /note="Xaa at position 12 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 14 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 14 is any hydrophobic 
amino acid. Y=a fluorogenic donor or acceptor 
radical which follows Xaa at position 14. V= 
an aminoalkylcarboxylic acid which may or may 
not be inserted between Y and Xaa at position 
14." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
GluArgAlaGlnAlaGlyValXa aAsnAlaSerXaaArgXaa 
1510 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor which 
precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at posiiton 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note="Xaa at position 11 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /note="Xaa at position 13 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 14 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 14. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
14." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
ArgAlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaaAla 
1510 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorgenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /note="Xaa at position 12 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A ) NAME/KEY: Modified-site 
(B) LOCATION: 14 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 14. V=an 
aminoalkylcarboxylic acid which may or may not be 
be inserted between Y and the amino acid at position 
14." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
Al aGlnAlaGlyValXaaAsnAlaSerXaaArgXaaAlaThr 
1510 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 5 
(D) OTHER INFORMATION: /note="Xaa at position 5 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is Ala or 
Cys." 
(i x) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note="Xaa at position 11 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 14 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amio acid at position 14. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
14." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
GlnAlaGlyValXaaAsnAlaSerXaaArgXaaAlaThrAla 
15 10 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is any 
hydrophobic amino acid." 
(i x) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /note="Xaa at position 13 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 15 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 15 is any hydrophobic 
amino acid. Y=a fluorogenic donor or acceptor 
radical which follows Xaa at position 15. V=an 
aminoalkylcarboxylic acid which may or may not 
be inserted between Y and Xaa at position 15." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
AlaGluArgAlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaa 
15 1015 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /note="Xaa at position 12 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 14 
(D) OTHER INFORMATION: /note= "Xaa at position 14 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 15 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 15. V=an 
aminoalkylcarboxylic acid which may or may not 
be inserted between Y and the amino acid at position 
15." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
GluArgAlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaaAla 
151015 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note="Xaa at position 11 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /note="Xaa at position 13 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 15 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 15. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
15." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
ArgAlaGlnAlaGlyValXa aAsnAlaSerXaaArgXaaAlaThr 
151015 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note="Xaa at position 6 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is Ala 
or Cys." 
(i x) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /note="Xaa at position 12 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 15 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 15. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
15." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
AlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaaAlaThrAla 
15 1015 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D ) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 14 
(D) OTHER INFORMATION: /note="Xaa at position 14 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 16 
(D) OTHER INFORMATION: /label=V- Y 
/note="Xaa at position 16 is any hydrophobic 
amino acid. Y=a fluorogenic donor or acceptor 
radical which follows Xaa at position 16. V= 
an aminoalkylcarboxylic acid which may or may 
not be inserted Y and Xaa at position 16." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
ValAlaGluArgAlaGlnAlaGlyValXaaAs nAlaSerXaaArgXaa 
151015 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note="Xaa at position 9 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /note="Xaa at position 13 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 15 
(D) OTHER INFORMATION: /note="Xaa at position 15 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 16 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 16. V= an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
16." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
AlaGluArgAlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaaAla 
15 1015 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 12 
(D) OTHER INFORMATION: /note="Xaa at position 12 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 14 
(D) OTHER INFORMATION: /note="Xaa at position 14 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 16 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 16. V=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Y and the amino acid at position 
16." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
GluArgAlaGlnAlaGlyValXaaAsnAlaSerXaaArgXaaAlaThr 
151015 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z- W 
/note="Z=a fluorogenic donor or acceptor radical 
which precedes the amino acid at position 1. W=an 
aminoalkylcarboxylic acid which may or may not be 
inserted between Z and the amino acid at position 
1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note="Xaa at position 11 is Ala 
or Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /note="Xaa at position 13 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 16 
(D) OTHER INFORMATION: /label=V- Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 16. V=an 
aminoalkylcarboxylic which may or may not be 
inserted between Y and the amino acid at position 
16." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
ArgAlaGlnAlaG lyValXaaAsnAlaSerXaaArgXaaAlaThrAla 
151015 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
-MF,300 (ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z 
/note="Xaa at position 1 is Gly or 4Abu or Acp or 
5- aminopentanoic acid or 7-aminoheptanoic acid. 
Z=a fluorogenic donor or acceptor radical 
which precedes Xaa at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /note="Xaa at position 4 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note="Xaa at position 8 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note="Xaa at position 10 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /label=Y 
/note="Y=a fluorogenic donor or acceptor radical 
which follows the amino acid at position 11." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
XaaGlyValXaaAsnAlaSerXaaArgXaaAla 
1510 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z 
/note="Xaa at position 1 is 4Abu. 
Z=4-(4'- dimethlylaminophenylazo)benzoyl 
(DABCYL) which precedes Xaa at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /label=Y 
/note= 
"Y=5-[(2-aminoethyl)amino]naphthalene-1-sulfonic 
acid (EDANS) which follows the amino acid at 
position 11." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
XaaGlyValValAsnAlaSerAlaArgLeuAla 
1510 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z 
/note="Xaa at position 1 is Gly or 4Abu or Acp 
or 5- aminopentanoic acid or 7-aminoheptanoic acid. 
Z=a fluorogenic donor or acceptor radical 
which precedes Xaa at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note="Xaa at position 3 is any 
hydrophobic amino acid." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note="Xaa at position 7 is Ala or 
Cys." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /label=Y 
/note="Xaa at position 9 is any hydrophobic amino 
acid. Y=a fluorogenic donor or acceptor radical 
which follows Xaa at position 9." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
XaaValXaaAsnAlaSerXaaArgXaa 
15 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1 
(D) OTHER INFORMATION: /label=Z 
/note="Xaa at position 1 is 4Abu. 
Z=4-(4'- dimethylaminophenylazo)benzoyl 
(DABCYL), which precedes Xaa at position 1." 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /label=Y 
/note="Y=5-[(2-aminoethyl)amino]naphthalene-1-sulfonic 
acid which follows amino acid at position 9." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
XaaValValAsnAlaSerAlaArgLeu 
15 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
ValAlaGluArgAlaGlnAlaGlyValValAsnAlaSerCysArgLeu 
1 51015 
AlaThrAla 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
( A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /label=Y 
/note="Y=5-[(2-aminoethyl)amino]naphthalene-1-sulfonic 
acid which follows amino acid at position 4." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
SerAlaArgLeu