The preparation of fluorescence-quenched probes for use in the characterization of human factor Xa substrate binding domains

Article abstract of DOI:10.3390/90600427

The preparation and characterization by LCMS of a library of 55 fluorescence-quenched peptides is described. The peptides bear a terminal anthranilamide fluorophore and a penultimate 2,4-dinitrophenyl-L-lysine quencher, and will be used to probe the subst

Full text of DOI:10.3390/90600427

Molecules 2004, 9, 427-439
molecules
ISSN 1420-3049
http://www.mdpi.org
The Preparation of Fluorescence-Quenched Probes for Use in
the Characterization of Human Factor Xa Substrate Binding
Domains
1
2
2,3,*
Karen M. Bromfield , Julia Cianci and Peter J. Duggan
1
Centre for Green Chemistry, Monash University, Clayton, Victoria, 3800. Australia.
School of Chemistry, Monash University, Clayton, Victoria, 3800. Australia.
CSIRO Molecular Science, Private Bag 10, Clayton South, Vic 3169, Australia; Phone: +61-3-9545
2
3
2
560; Fax: +61-3-9545 2446.
*
Author to whom correspondence should be addressed; e-mail peter.duggan@csiro.au
Received: 4 March 2004 / Accepted: 15 March 2004 / Published: 31 May 2004
Abstract: The preparation and characterization by LCMS of a library of 55 fluorescence-
quenched peptides is described. The peptides bear a terminal anthranilamide fluorophore
and a penultimate 2,4-dinitrophenyl-L-lysine quencher, and will be used to probe the
substrate binding domain of the human blood coagulation enzyme, factor Xa.
Keywords: Solid phase peptide synthesis, parallel synthesis, fluorescence-quenched
substrates, protease, factor Xa.
Introduction
Factor Xa is an arginine-specific serine protease that operates at the convergence of the extrinsic
and intrinsic blood coagulation pathways [1]. The prothrombinase complex, which consists of factors
Xa and Va, phospholipid and calcium, is responsible for limited proteolysis of prothrombin to give
thrombin. These facts, along with factor Xa’s relatively specific physiological role [2], make it a prime
target for the development of new selective anticoagulants. Intimate knowledge of the protein

Molecules 2004, 9
428
sequences most preferred by factor Xa provides insight into the enzyme’s physiological role, its
interaction with natural inhibitors and allows more effective design of selective anticoagulants. Most
peptidic inhibitors of serine proteases have been designed to associate with the substrate binding sites
on the N-terminal (“non-prime”) side of the cleavage site [3]. However, inhibitors that associate with
the enzyme on both sides of the catalytic serine [4] have the potential to bind more strongly and be
more selective between related proteases.
We have previously probed the selectivity of bovine factor Xa for the first three substrate amino
acid residues on the “prime” side of the cleavage site, P ’, P ’ and P ’[5]. Fluorescence quenched
1
2
3
peptide substrates [6] spanning both sides of the scissile bond (P -P ’) were used to investigate such P’
1
1
specificities in a similar way to that used for other proteases [7,8]. These probes contain an N-terminal
-aminobenzoyl (Abz) group and a penultimate 2,4-dinitrophenyl (Dnp) derivitized lysine (e.g. 1,
Figure 1). The fluorescence of the N-terminal Abz group (λ = 325 nm, λ = 414 nm) is quenched in
2
ex
em
the intact peptides by the Dnp group through intramolecular resonance energy transfer [6]. Peptide
hydrolysis by factor Xa relieves this intramolecular fluorescence quenching and the resulting increase
in fluorescence is proportional to the concentration of the released fluorophoric fragment (2). This
enables determination of kinetic parameters by monitoring the change in fluorescence intensity with
time.
Figure 1. Intramolecular quenching of the Abz fluorophore by the Dnp-lysine residue
in the peptidic probes is relieved on cleavage, adjacent to the arginine
residue, by the protease, factor Xa.
Results and Discussion
While peptidic probes of the type mentioned above have been sited a number of times in the
biological literature, a detailed description of the preparation and characterization of such compounds,
2
6
as well as their precursors, N-(Boc)-anthranilic acid and N -Fmoc-N -dinitrophenyl-L-lysine, has yet to
appear. In this article, we provide a comprehensive description of the preparation of a library of
peptidic fluorescence-quenched probes that are being used to probe the “prime-side” substrate

Molecules 2004, 9
429
specificities of human factor Xa. The biological results obtained with these probes will be published at
a later date.
Substrates suitable for the present study were prepared using parallel solid phase peptide synthesis
techniques incorporating Fmoc/HBTU/TFA chemistry (see General Experimental section for a list of
abbreviations used in this article) and is summarised in Scheme 1. Briefly, this involved building the
peptides on a solid phase bearing a HMPA linker, with aspartate attached through the side chain
carboxylic acid and Fmoc-protected on the nitrogen, and with the α-carboxylic acid protected with a
tert-butyl group.
Scheme 1.

Molecules 2004, 9
430
The Fmoc protecting group was removed with piperidine and the next amino acid, with Fmoc and
side chain protection, was attached with HBTU activation. The terminal Fmoc group was then
cleaved, again with piperidine, and the two-step process was repeated with the next protected amino
acid until the desired sequence was obtained. The peptide was then cleaved from the resin, and the
amino acid side chain protecting groups removed, by treatment with TFA in the presence of anisole
and EDT. Where coupling steps were found to be low yielding, a “double coupling” was performed, in
which the solid phase was treated twice with the activated amino acid prior to Fmoc removal. In some
cases, it was also necessary to re-treat the cleaved peptides with TFA to remove the last traces of tert-
butyl side-chain protecting groups. This overall strategy typically allowed the production of peptides
in >60% yield and >90% purity (see Tables 2-4).A library of 55 fluorescence-quenched peptides was
prepared with the sequence being; Abz-Ile-Glu-Gly-Arg-P ’-P ’-P ’-Ser-Lys(Dnp)-Asp-OH (1). The
1
2
3
most common naturally occurring amino acids, excluding cysteine, were substituted at the P ’, P ’ or
1
2
P ’ positions while the remainder of the sequence was unchanged, with alanine being used as the
3
default residue for the P
1
’-P ’ positions.
3
Conclusions
We have presented a detailed description of the preparation and characterization of a library of 55
fluorescence-quenched peptidic probes for use in determination of the “prime-side” substrate
specificities of human factor Xa. More than 87% of these peptides were obtained in >60% yield and
all were obtained in >90% purity, as judged by LCMS.
Acknowledgements
This work was supported by the National Heart Foundation, the Australian Research Council and
Monash University. K.M.B. is a recipient of a Monash Research Scholarship. Ian James of
Mimotopes Pty Ltd is thanked for helpful advice.
Experimental
General
Abbreviations: Abz: 2-aminobenzoyl; BOP: benzotriazol-1-yl-oxy-tris-(dimethylamino)
phosphonium hexafluorophosphate; Dnp: 2,4-dinitrophenyl; EDT: 1,2-ethanedithiol; HBTU: 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorphosphate; HMPA: hydroxymethylphenoxy-
acetic acid; HOBt.H O : hydroxybenzotriazole monohydrate; LCMS: Liquid chromatography mass
2
spectrometry; NMM: N-methylmorpholine; Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Trt: trityl.
All N-Fmoc-amino acids, HBTU and HOBt.H O were obtained from Novabiochem (Merck). All other
2
reagents were obtained from Sigma-Aldrich. Solvents were used as supplied (AnalR grade) unless
otherwise stated. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX

Molecules 2004, 9
431
1
13
3
00MHz spectrometer ( H-, 300 MHz; C-, 75 MHz). The spectra were run at ambient temperature
using the appropriate deuterated solvent with all chemical shifts referenced to the residual protonated
solvent peak. Chemical shifts (δ) are reported in parts per million (ppm), followed by their
multiplicities, coupling constants (J Hz) where appropriate, intensities and assignments. Infrared
spectra were recorded using a Perkin Elmer 1600 Series Fourier Transform Spectrophotometer. The
spectra of oils were recorded as thin films (neat) mounted on NaCl plates, whereas solids were
recorded as either nujol mulls on NaCl plates or as KBr disks. Principal diagnostic absorptions are
-1
reported in wavenumbers (cm ) followed by assignment were appropriate. All mass spectra used are
electrospray ionisation (ESI ) unless otherwise stated. High resolution mass spectra were recorded on a
Bruker BioApex 47e Fourier Transform mass spectrometer. Samples were dissolved in methanol or 1:4
dichloromethane - methanol, and ionized using an ESI source. Melting points of solid compounds were
measured on a Reichert hot stage melting point apparatus and are uncorrected. Flash chromatography
refers to wet-packed columns with Merck silica gel 60 (0.040-0.063 mm, 230-400 mesh ASTM) as the
stationary phase. Pre-absorption was carried out using the same silica. Air pressure was applied to
columns to give an approximate flow rate of 2.5 mL of eluent per minute. Thin layer chromatography
was performed on silica coated aluminium-backed sheets (silica gel 60/ F ). Compounds were
2
54
routinely visualised under short wavelength (254 nm) ultraviolet light. Unless otherwise stated, LCMS
analysis was undertaken by Phillip Holt (Centre for Green Chemistry) with the following setup:
Equipment; Gilson 215 Liquid handler/autosampler, Gilson 819 injector module, Gilson 811 dynamic
mixer 700 µL chamber, Gilson 306 Pumps with 50.SC heads, Agilent 1100 series G1315A Diode
array detector, Column Alltima C18 150 x 4.6 mm, 5 µm particle size, Alltech Australia, Mass
Spectrometer; Micromass ZMD from Waters Australia, software; Micromass MassLynx version 3.5.
LC Method: Dry samples were dissolved in 300 µL of 30% acetonitrile/water, of which 20 or 40 µL
was injected into the HPLC instrument. HPLC Solvents: A: 0.5% Formic acid in Milli Q water, B:
0
1
.5% Formic acid in 80% acetonitrile, 20% Milli-Q water. Flow rate of 1 mL/min and gradient 10-
00% B over 10 minutes. MS Method: (Electrospray Ionization +ve Mode) capillary 3kV, cone 30V,
source temp 80°C, de-solvation temperature 150°C, desolvation gas 200L/hr, cone gas 50L/hr.
Scanning; 350 to 800 amu in 0.8 seconds, 0.1 seconds interscan delay.
2
6
N -Fmoc-N -dinitrophenyl-L-lysine
Following the procedure of Anastasi et al. [9], diisopropylethylamine (1.5 mL, 8.6 mmol) was
6
added to commercial N -dinitrophenyl-L-lysine (1.50 g, 4.3 mmol) in 1:1 acetonitrile/water (22 mL).
The resulting solution was stirred vigorously at 0°C and Fmoc-succinimidyl carbonate (1.50 g, 4.4
mmol) was added over 25 minutes. After a further 30 minutes, the reaction mixture was allowed to
warm to room temperature and left to stir for 20 hours. The reaction mixture was then diluted with
ethyl acetate (100 mL) and the organic phase washed with 10% citric acid (100 mL), water (3 x 100
mL) and saturated NaCl (100 mL). The organic extract was dried with MgSO
4
and the solvent
evaporated under vacuum to give a yellow oil. Flash column chromatography eluting with

Molecules 2004, 9
432
9
.5:0.25:0.25 chloroform/methanol/acetic acid was followed by co-evaporation with toluene and
trituration with hexane to give the title compound as a yellow solid, (2.11 g, 92%); mp: 79-82 °C, lit.
8
1
1-83 °C [9]; H-NMR (300 MHz, CDCl ): δ 9.09 (d, J = 2.6 Hz, 1H, (NO ) -Ar-H), 8.50 (br s, 1H,
3
2 2
(
7
9
NO
2
)
2
-Ar-NH), 8.22 (dd, J = 9.6, 2.5 Hz, 1H, (NO
2
)
2
-Ar-H), 7.75 (d, J = 7.3 Hz, 2H, Fluor-H), 7.61-
.54 (m, 2H, Fluor-H), 7.39 (t, J = 7.2 Hz, 2H, Fluor-H), 7.30 (t, J = 7.4 Hz, 2H, Fluor-H), 6.86 (d, J =
.6 Hz, 1H, (NO ) -Ar-H), 5.38 (d, J = 7.5 Hz, 1H, OCONH), 4.56-4.43 (m, 1H, H2), 4.42 (d, J = 6.7
2
2
Hz, 2H, OCH
2
CH), 4.20 (t, J = 6.8 Hz, 1H, OCH CH), 3.42-3.35 (m, 2H, H6), 2.06-1.93 (m, 2H, H3),
2
1
3
1
1
2
.90-1.73 (m, 2H, H5), 1.65-1.51 (m, 2H, H4); C-NMR (75 MHz, d -DMSO): δ 173.4, 155.6, 147.6,
43.3, 140.2, 134.1, 129.1, 129.4, 127.1, 126.5, 124.7, 123.1, 119.6, 114.7, 65.1, 53.2, 46.2, 42.1, 29.9,
7.1, 22.5; IR (nujol): 3436 s, 3370 s, 1733 s, 1692 m, 1619 s, 1589 m, 1520 s, 1503 m, 1337 s; MS:
6
+
+
m/z 557.3 (MNa , 100%), 535.3 (MH , 81%), 357.3 (C H N O , 34%), 313.2 (C H N O , 47%);
1
3
17
4
8
12 17
4
6
+
HRMS: m/z [MNa , C H N O .Na] calcd 557.1648; found 557.1643.
2
7
26
4
8
2
-Amino(tert-butoxycarbonyl)benzoic acid
Following the method of Mougenot and Marchand-Brynaert [10] NaOH (0.65 g, 16.5 mmol), in
water (16 mL) and tert-butanol (22 mL), was added to anthranilic acid (2.05 g, 14.9 mmol). Di-tert-
butyl dicarbonate (3.23 g, 14.8 mmol) was then added with vigorous stirring over 15 minutes. The
reaction was left to stir at room temperature for 17 hours after which time it was cooled to 0°C and
acidified to pH 2-3 with citric acid (10% w/v). The reaction mixture was then extracted with ether (2 x
1
00 mL then 2 x 50 mL) and the combined organic extracts were dried over MgSO and the solvent
4
evaporated under vacuum. The residue was recrystallised from dichloromethane and methanol to give
Boc protected anthranilic acid as white needles (2.16 g, 61%); mp: 150-152 °C, lit. 155-157 °C [11];
1
H-NMR (300 MHz, CDCl
3
): δ 10.03 (s, 1H, NH), 8.47 (dd, J = 8.6, 0.8 Hz, 1H, H6), 8.09 (dd, J =
8
.0, 1.6, Hz, 1H, H3), 7.57 (dt, J = 7.9, 1.6 Hz, 1H, H4), 7.04 (dt, J = 7.6, 1.1 Hz, 1H, H5), 1.55 (s, 9H,
1
3
t-Bu); C NMR (75 MHz, CDCl
3
): δ 173.0, 152.9, 143.1, 135.7, 132.1, 121.5, 119.2, 113.5, 81.0,
+
+
2
8.5; IR (nujol): 3318 s, 1728 s, 1670 s; MS: m/z 260.3 (MNa , 38%), 204.2 (MNa -tBu, 16%), 182.2
+
+
+
(
MH -tBu, 9%), 138.2 (MH -Boc, 100%); HRMS: m/z [MNa , C H NO .Na] calcd 260.0899; found
1
2
15
4
2
60.0894.
General Procedure for the Synthesis of Individual Fluorescence Quenched Peptides
Peptides were prepared following the Mimotopes procedures [12] with some modifications. These
modifications allowed the use of HBTU as the activating agent, in place of BOP. All solvents/reagents
were dried and purified as specified [12]. Mimotopes Series I crowns bearing HMPA linkers and an
Fmoc protected Asp residue (which formed the C-terminus of the completed peptides) were the
starting point of the synthesis of all peptides. These crowns conveniently fit into 1.6 mL Eppendorf
tubes, which were used as reaction vessels for the Fmoc-deprotection, coupling and washing steps.

Molecules 2004, 9
433
Fmoc Deprotection
Each crown was immersed in 20% piperidine/DMF (750 µL) for 20 minutes. The crown was then
washed several times, firstly with DMF (750 µL) for 2 minutes, then with methanol (3 x 750 µL) also
for 2 minutes each. The crowns were then allowed to dry at room temperature on a watchglass for 30
minutes.
Amino Acid and Activation Solutions
The desired L-amino acid with N-Fmoc protection (0.116 mmol, see Table 1 for mass) was
dissolved in DMF (320 µL). A second ‘activating’ solution containing HBTU (44.2 mg, 0.116 mmol),
HOBt.H O (17.8 mg, 0.116 mmol) and NMM (19.2 µL, 0.175 mmol) in DMF (445 µL to give final
2
volume of 510 µL) was also prepared.
Amino Acid Coupling
The Fmoc-L amino acid solution (310 µL of the 320 µL) was added to the activating solution (490
µL of the 510 µL) to give a coupling solution (800 µL) containing Fmoc-L-amino acid (140 mM),
HBTU (140 mM), HOBt.H O (140 mM) and NMM (210 mM). After 2 minutes, the dry deprotected
2
crown was immersed in the coupling solution and left to stand for at least 2 hours. The crowns were
then removed and washed with methanol (750 µL) for 5 minutes, dried at room temperature for 2
minutes and then washed with DMF (750 µL) for 5 minutes. The crown was then subjected to repeated
cycles of the above deprotection and coupling steps to give the amino acid sequence required. An
alternative post-coupling washing procedure was used when the next cycle could not be started
immediately. This involved the crown being washed with DMF (750 µL) for 5 minutes, dried for 2
minutes and then washed with methanol (750 µL) for 5 minutes. After drying, the crown was stored at
room temperature in a covered petri dish until needed.
Side Chain Deprotection and Cleavage
After coupling of the final residue, Abz in this case, the crown was washed with DMF (750 µL) for
minutes, then with methanol (2 x 750 µL) for 2 minutes each and allowed to air dry for 30 minutes.
2
The crown was then placed in a 10 mL plastic centrifuge tube containing the cleavage solution, which
consisted of TFA (1.24 mL), EDT (37.5 µL), anisole (75 µL), thioanisole (75 µL) and water (75 µL).
After 2.5 hours, the crown was then removed and washed with TFA (0.5 ml). The TFA wash was then
added to the peptide solution that remained in the centrifuge tube and the mixture was concentrated to
~
10% volume under a flow of nitrogen gas (~2 hours).

Molecules 2004, 9
434
Trituration
To the concentrated peptide solution was added a solution of 1:2:0.1% ether/hexane/
mercaptoethanol (8 mL) and the mixture was shaken. The suspension was placed in a freezer (approx.
-
20°C) for 30 minutes, centrifuged and the supernatant decanted from the solid. Additional 1:2
ether/hexane (4 mL) solution was added and again shaken, cooled, centrifuged and the supernatant
removed. The resulting pellet was dried under nitrogen, suspended in H O and freeze-dried to give the
2
desired peptide typically in quantitative yield. The purity of the product was determined using LCMS.
Table 1: Mass Required of Fmoc and Side-chain Protected Amino Acids
Amino Acid Code
Protecting
Group
−
Mass (mg)A
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
36.0
76.6
69.0
47.6
67.7
70.6
49.2
34.4
71.7
40.9
40.9
54.2
42.9
44.8
39.0
44.3
46.0
60.9
53.1
39.2
61.8
27.4
Pmc
Trt
OtBu
Trt
Trt
OtBu
−
Trt
−
Ile
Leu
Lys
Met
Phe
Pro
−
Boc
−
−
−
Ser
tBu
tBu
Boc
tBu
−
Thr
Trp
Tyr
Val
Lys(Dnp)
−
B
Abz
−
A
B
0
.116 mmol, Nitrogen protected with Boc rather than Fmoc.

Molecules 2004, 9
Synthesis of P ’-P ’ Fluorescence Quenched Substrate Library
435
1
3
The factor Xa P
1
’, P
2
’ and P ’ library consisted of 55 peptides. All had the sequence Abz-Ile-Glu-
3
Gly-Arg-P ’-P ’-P ’-Ser-Lys(Dnp)-AspOH were P ’, P ’ and P ’ were varied individually using
1
2
3
1
2
3
alanine as the default residue. The procedure for the synthesis of these probes followed the “General
Procedure for the Synthesis of Individual Fluorescence Quenched Peptides” (above) with the following
exceptions: Following the coupling of the P ’ amino acid, and before Fmoc removal, the crowns were
1
subjected to a second immersion in fresh coupling solution (containing the activated P ’ amino acid)
1
followed by the appropriate washes, before the next deprotection and coupling cycles were undertaken.
In addition, instead of individual eppendorf tubes, a reaction tray containing 96 wells was utilized in
conjunction with a pin holder on which crowns attached to stems were mounted. All Fmoc-
deprotection, coupling, washes and drying was undertaken with the crowns attached to the pin holder.
This allowed efficient synthesis of the large number of peptides in parallel. Side chain deprotection
and cleavage was still performed in individual centrifuge tubes. To allow for the smaller reaction
vessels in the 96-well plate, quantities used differed as follows: The deprotection and washing steps all
used 700 µL. The amino acid solution contained 0.102 mmol (rather than the 0.116 mmol quantities
listed in Table 1) of the required protected amino acid in DMF (280 µL). The activating solution
contained HBTU (38.9 mg, 0.102 mmol), HOBt.H O (15.7 mg, 0.102 mmol) and NMM (17 µL, 0.155
2
mmol) in DMF (405 µL resulting in a final volume of 450 µL). The amino acid solution (270 µL of the
2
80 µL) was added to the activating solution (430 µL of the 450 µL) to give the coupling solution (700
µL) containing Fmoc-L-amino acid (140 mM), HBTU (140 mM), HOBt.H O (140 mM) and NMM
210 mM), to which the crown was added. The peptidic products were individually cleaved from the
2
(
crowns and triturated in 10 mL plastic centrifuge tubes in the same way as that described above for the
preparation of individual peptides. The yields obtained are shown in Tables 2-4. Characterisation by
LCMS in most cases identified the major constituent to be the desired peptide, as indicated by the
retention time and m/z reported in the Tables. For some peptides, however, a second peak, which was
identified as the desired peptide with a t-butyl group still attached was also observed in the LCMS
chromatogram. In these cases, a second treatment with the TFA cleavage solution, followed by
trituration and freeze-drying, completed the deprotection of the peptides ensuring that they were
obtained in greater than 90% purity. In some cases, initial LCMS analysis also revealed the presence of
another ion, which was apparently due to fragmentation of the peptide in the mass spectrometer. These
latter ions corresponded to the doubly protonated peptide having lost the 2 N-terminal residues (Abz-
Ile).

Molecules 2004, 9
436
Table 2: Characterization of P ' Probe Library
1
Relative
Peptide
Code
Yield
(%)
MW
Retention
Time (min)
Peak Area
m/z E
674.0
681.0
690.1
644.9
457.2
Assignment
A
B
C
D
(%)
+
KBLD
1346.32
1360.35
1378.41
1288.28
1368.37
84
76
96
42
49
9.17
9.08
9.39
100
(M+2H )/2
+
KBL1E
KBL1F
KBL1G
KBL1H
>90
100
100
100
(M+2H )/2
+
(M+2H )/2
F
F
G
+
11.6
(M+2H )/2
G
+
10.70
(M+3H )/3
+
6
5
85.0 (80 %) (M+2H )/2
68.8 (35 %) (M-
+
2
32+2H )/2
72F
78
12.2G
8.73
100
100
673.1
454.3
(M+2H )/2
+
KBL1I
KBL1K
1344.39
1359.40
+
(M+3H )/3
+
6
5
80.6 (45 %) (M+2H )/2
64.5 (20 %) (M-
+
2
32+2H )/2
46F
73
78
69
12.18 G
9.21
8.84
100
100
>90
>90
>90
>90
673.0
682.3
673.7
665.2
680.5
463.7
(M+2H )/2
+
KBL1L
KBL1M
KBL1N
KBL1P
KBL1Q
KBL1R
1344.39
1360.43
1345.33
1328.35
1359.36
1387.42
+
(M+2H )/2
+
(M+2H )/2
+
9.01
11.64
(M+2H )/2
F
G
+
55
(M+2H )/2
+
92
8.66
(M+3H )/3
+
6
5
94.6 (60 %) (M+2H )/2
78.6 (40 %) (M-
+
2
32+2H )/2
54F
11.75
11.70
G
100
100
100
100
>90
660.0
667.1
666.2
709.8
698.0
(M+2H )/2
+
KBL1S
KBL1T
KBL1V
KBL1W
KBL1Y
1318.31
1332.34
1330.36
1417.44
1394.41
F
G
+
62
(M+2H )/2
+
64
91
79
9.16
9.38
9.27
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
A
B
For example: KBL1E contains a Glu (E) residue at P
1
’, Ala at P
2
’ and P
3
’; Isolated yield
C
based on approximate crown loading of 6.1 µmol. Q = quantitative;
See General
D
Experimental section for LCMS conditions; Derived from total ion current chromatogram;
E
F
Relative Abundance of 100% unless otherwise stated; After second TFA treatment, yield
G
calculated from the original crown loading of 6.1 µmol; LCMS gradient run was extended
from the typical 10-100% solvent B over10 minutes to 10-100% solvent B over 15 minutes.

Molecules 2004, 9
437
Table 3: Characterization of P ' Probe Library
2
Retention
Time
(min)
Relative
Peptide
Code
Yield
(%)
Peak Area
A
B
C
D
m/z E
MW
(%)
Assignment
+
KBL2A
KBL2D
KBL2E
KBL2F
KBL2G
KBL2H
1302.31 92
1346.32
9.28
9.16
9.23
9.50
9.30
8.77
>90
>90
>90
>90
>90
100
652.0
674.2
681.2
690.2
645.2
457.3
(M+2H )/2
+
Q
(M+2H )/2
+
1360.35 92
1378.41 93
1288.28 62
1368.37 78
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
+
(M+3H )/3
+
6
5
85.2 (55 %) (M+2H )/2
69.0 (20 %) (M-232+2H )/2
+
+
KBL2I
KBL2K
1344.39 73
9.34
8.77
100
100
673.2
453.7
(M+2H )/2
+
1359.40
Q
(M+3H )/3
+
6
5
80.7 (95 %) (M+2H )/2
64.6 (25 %) (M-232+2H )/2
+
+
KBL2L
KBL2M 1360.43
KBL2N
KBL2P
KBL2Q
KBL2R
1344.39 79
9.43
9.36
9.17
9.19
9.17
8.92
>90
100
100
>90
100
100
673.3
681.9
673.7
665.2
680.4
694.6
(M+2H )/2
+
Q
(M+2H )/2
+
1345.33 67
1328.35
(M+2H )/2
+
Q
(M+2H )/2
+
1359.36 93
1387.42 98
(M+2H )/2
+
(M+2H )/2
+
5
78.4 (63 %) (M-232+2H )/2
1318.31 89F
1332.34 87
1330.36 86F
9.12
9.16
9.23
9.47
9.19
100
>90
>90
100
100
660.0
667.1
665.9
709.7
698.1
(M+2H )/2
+
KBL2S
KBL2T
KBL2V
KBL2W 1417.44 95
KBL2Y
+
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
1394.41 92F
(M+2H )/2
+
A
B
For example: KBL2E contains a Glu (E) residue at P
2
’, Ala at P
1
’ and P
3
’; Isolated yield
C
based on approximate crown loading of 6.1 µmol. Q = quantitative;
See General
D
Experimental section for LCMS conditions; Derived from total ion current chromatogram;
E
F
Relative Abundance of 100% unless otherwise stated; After second TFA treatment, yield
calculated from the original crown loading of 6.1 µmol.

Molecules 2004, 9
438
Table 4: Characterization of P ’ Probe Library
3
Relative
Peptide
Code
Yield
(%)
Retention
Time (min)
MW
Peak Area
m/z E
674.1
681.0
690.1
654.1
457.3
Assignment
A
B
C
D
(
%)
+
KBL3D
KBL3E
KBL3F
KBL3G
KBL3H
1346.32
1360.35
1378.41
1288.28
1368.37
91
86
86
82
91
9.30
9.17
9.47
8.94
8.83
>90
100
100
100
100
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
+
(M+3H )/3
+
6
5
85.1 (40 %) (M+2H )/2
69.0 (30 %) (M-
+
2
32+2H )/2
+
KBL3I
KBL3K
1344.39
1359.40
68
95
9.36
8.84
100
>90
673.0
454.3
(M+2H )/2
+
(M+3H )/3
+
6
5
80.7 (55 %) (M+2H )/2
64.6 (40 %) (M-
+
2
32+2H )/2
+
KBL3L
KBL3M
KBL3N
KBL3P
KBL3Q
KBL3R
1344.39
1360.43
1345.33
1328.35
1359.36
1387.42
61
61
96
89
94
93
9.41
9.34
9.01
9.12
9.06
8.75
>90
100
100
100
100
>90
673.0
682.0
673.5
665.2
680.7
463.7
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
+
(M+3H )/3
+
6
5
94.8 (50 %) (M+2H )/2
78.6 (25 %) (M-
+
2
32+2H )/2
59F
11.55
11.81
G
100
100
100
100
100
660.0
667.1
666.2
709.8
698.2
(M+2H )/2
+
KBL3S
KBL3T
KBL3V
KBL3W
KBL3Y
1318.31
1332.34
1330.36
1417.44
1394.41
F
G
+
54
(M+2H )/2
+
74
89
75
9.21
9.39
9.17
(M+2H )/2
+
(M+2H )/2
+
(M+2H )/2
A
B
For example: KBL3E contains a Glu (E) residue at P
3
’, Ala at P
1
’ and P
2
’; Isolated yield based on
C
approximate crown loading of 6.1 µmol. Q = quantitative; See General Experimental section for
LCMS conditions; D Derived from total ion current chromatogram; Relative Abundance of 100%
E
F
unless otherwise stated; After second TFA treatment, yield calculated from the original crown
loading of 6.1 µmol; G LCMS gradient run was extended from the typical 10-100% solvent B over10
minutes to 10-100% solvent B over 15 minutes.

Molecules 2004, 9
439
References
1
2
3
.
.
.
Davie, E. W.; Fujikawa, K.; Kisiel, W. The coagulation cascade - initiation, maintenance, and
regulation. Biochemistry 1991, 30, 10363-10370.
Powers, J. C.; Harper, J. W. In Proteinase Inhibitors, 2 ed.; Barrett, A. J.; Salvesen, G., Eds.;
nd
Elsevier: Amsterdam, 1986; Vol. 12; pp 55.
Lottenberg, R.; Hall, J. A.; Pautler, E.; Zupan, A.; Christensen, U.; Jackson, C. M. The action of
factor Xa on peptide p-nitroanilide substrates: Substrate selectivity and examination of hydrolysis
with different reaction conditions. Biochim. Biophys. Acta 1986, 874, 326.
4
5
.
.
Walker, B.; Lynas, J. F. Strategies for the inhibition of serine proteases. Cell. Mol. Life Sci., 2001,
5
8, 596-624 and references therein.
Ludeman, J. P.; Pike, R. N.; Bromfield, K. M.; Duggan, P. J.; Cianci, J.; Le Bonniec, B.;
Whisstock, J. C.; Bottomley, S. P. Determination of the P’ , P’ and P’ subsite-specificity of
1
2
3
factor Xa. Int. J. Biochem. Cell Biol., 2003, 35, 221-225.
Yaron, A.; Carmel, A.; Katchalski-Katzir, E. Intramolecularly quenched fluorogenic substrates for
hydrolytic enzymes. Anal. Biochem., 1979, 95, 228-235.
Le Bonniec, B. F.; Myles, T.; Johnson, T.; Knight, C. G.; Tapparelli, C.; Stone, S. R.
Characterization of the P-2' and P-3' specificities of thrombin using fluorescence-quenched
substrates and mapping of the subsites by mutagenesis. Biochemistry, 1996, 35, 7114-7122.
Marque, P.-E.; Spuntarelli, R.; Juliano, L.; Aiach, M.; Le Bonniec, B. F. The role of Glu(192) in
the allosteric control of the S-2 ' and S-3 ' subsites of thrombin. J. Biol. Chem., 2000, 275, 809-
6
7
.
.
8
.
.
8
16.
9
1
Anastasi, A.; Knight, C.G.; Barrett, A.J. Characterization of the bacterial metalloendopeptidase
pitrilysin by use of a continuous fluorescence assay. Biochem. J., 1993, 290, 601-607.
0. Mougenot, P.; Marchand-Brynaert, J. Reactivity assays of surface hydroxyl chain ends of
poly(ethylene terephthalate) (PET) film and membranes using original H-3 and fluorine-labeled
derivatization reagents Macromolecules, 1996, 29, 3552-3559.
1
1
1. Heyman, D. A. Preparation of isatoates from isatoic anhydride. J. Heterocyclic Chem., 1978, 15,
1
131-1136.
2. Chiron Technologies, Multipin Peptide Synthesis Kit, Software Manual, Chiron Technologies,
Pty. Ltd. (now Mimotopes Pty. Ltd.), 1997.
Sample availability: Contact authors
2004 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.
©