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2293
and HOBt). Unreacted amino groups on the resin were
then capped with acetic anhydride.
The strategy behind the tetrapeptide PS-SCL is the gen-
eration of three sublibraries, one library for each sequence
position evaluated (P2, P3, and P4). Each sublibrary is
composed of one pot for each amino acid evaluated. In
the current study, 20 amino acids were investigated
resulting in 20 pots per sublibrary. In each pot, the residue
at one sequence position (e.g., P2) is known. Using a P2
sublibrary as an example, this is achieved by dividing 9
into 20 pots and coupling a dierent amino acid in each
pot. In the next coupling sequence, an isokinetic mix-
ture of the 20 amino acids is used to generate all possi-
ble 20 tripeptides in each pot in equal proportions. For
the ®nal coupling, the same isokinetic mixture is used to
generate in each of the 20 pots all possible 400 tetra-
peptide substrates with a dierent, known P2 residue.
Following cleavage from the resin, each pot in the sub-
library is assayed against the enzyme and the optical
density (OD) measured. Since each pot contains all
possible tetrapeptides with a known P2 residue, those
pots displaying the highest OD contain substrates that
are most eciently cleaved and therefore the preferred
P2 residues. Similar analysis of the P3 and P4 sub-
libraries identi®es the preferred P3 and P4 residues.
Figure 1. MALDI-TOF mass spectra (re¯ectron mode, alpha-cyano
hydroxycinnamic acid matrix) of P3 sublibrary pots (a) Ac-O-Val-O-
Arg-Amc and (b) Ac-O-Asp-O-Arg-Amc.
Solid-phase synthesis of each sublibrary was accom-
plished using Fmoc protected single amino acids or an
isokinetic mixture of 20 amino acids, HATU coupling,
and piperidine deprotection. The loading was monitored
using the Kaiser test, with a negative test being obtained
before proceeding with the deprotection. The HATU
coupling conditions and proportions of amino acids in
the isokinetic mixture were those developed by Herman
et al.7 Following cleavage from the resin, the crude
material was isolated by solvent removal and subsequent
ether trituration. No further puri®cation was employed
so as to avoid selective removal of any peptides. Initially,
cleavage of the peptides from the resin using a standard
TFA:H2O:phenol:TIS (87.5:5:5:2.5) cocktail and a PEG
polystyrene resin aorded only a 5% yield of peptide
mixture. Changing to the AgroPoreTM-NH2 resin and
employing a stronger, TMSBr-based cleavage cocktail8
increased the yield to 50±70% (75±105 mg/pot).
Ac-O-Asp-O-Arg-Amc pots are 705 and 721, respec-
tively. The centers of distribution in Figure 1 are in
good agreement with these calculated values.
While the chemical characterization of the library sug-
gested that equimolar mixtures of peptides were present in
the pots analyzed, the most important and practical
means for validating the ®delity of the library was to
assay each sublibrary against an enzyme with known
substrate speci®city. Thrombin was chosen because it is
a trypsin-like serine protease of current medicinal inter-
est. In addition, thrombin has a varied subsite speci®city
that we felt would be ideal for validating the library: an
extremely strong preference for proline at P2; a rela-
tively broad speci®city at P3; and a preference for
hydrophobic residues at P4.9 The libraries were assayed
by incubating each sample with enzyme and measuring
the ¯uorescence.
We conducted a limited chemical characterization of the
library. The P3 sublibrary Ac-O-Val-O-Arg-Amc and
Ac-O-Asp-O-Arg-Amc pots were analyzed by means of
MALDI-TOF mass spectrometry. The mass spectral
data was assessed for the presence of three features: (1)
that the smallest (-G-V-G-) and largest (W-V-W-)
molecular weight peptides were present; (2) that the
center of the distribution for each of the two pots was
shifted from each other by 16, the dierence in mole-
cular weight between valine and aspartic acid; and (3)
that the molecular weight distribution was centered
around the average molecular weight. Figure 1 is a
comparison of the mass spectra for the -Val- and -Asp-
pots. As anticipated, the high and low molecular weight
compounds were present and the center of distribution
between each library was shifted by 16. The average
molecular weights for the Ac-O-Val-O-Arg-Amc and
The results against each sublibrary are depicted in Fig-
ure 2. In the P2 library, only ®ve amino acids demon-
strated any signi®cant hydrolysis by thrombin, all small
hydrophobic residues. Proline (P) was clearly preferred.
Consistent with the literature, a broad variety of resi-
dues were tolerated at P3, including basic, polar, and
both large and small hydrophobic residues. Proline,
which is a unique cyclic amino acid that imparts a turn
into the peptide backbone, was completely inactive at
the P3 position. Signi®cantly, valine (V), leucine (L) and
isoleucine (I) were preferred over phenylalanine (F) as
recently reported for peptidic chloromethyl ketone inhi-
bitors.10 Particularly interesting is the comparison of
aspartic acid (D) and its amide asparagine (N) with