Inhibition properties of free and conjugated leupeptin analogues

Article abstract of DOI:10.1002/2211-5463.12994

Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The specificity of leupeptin is most likely due to the Leu–Leu–Argininal sequence, and its C-terminal aldehyde group has been suggested to enhance the binding efficiency and to be essential for function. The terminal aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether the inhibitory function of leupeptin toward serine proteases is retained after oxidation or reduction of the aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced inhibitory properties as compared with the oxidized form. Based on these results, new synthetic leupeptin analogues, 6-aminohexanoic acid (Ahx)–Phe–Leu–Arg–COOH and Ahx–Leu–Leu–Arg–COOH, were prepared by solid-phase peptide synthesis using the Fmoc strategy. In these analogues, the N-terminal capping acetyl group was replaced with a 6-aminohexanoyl group to allow conjugation. The structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry. Determination of the inhibitory properties against trypsin (IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1) revealed that these further modified tripeptides were tight binding inhibitors to their target enzyme, similar to the naturally occurring leupeptin, with K_i values generally in the micromolar range. The Ahx–Phe–Leu–Arg–COOH analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads. All conjugates exhibited inhibitory activity in the same range as for the free peptides.

Full text of DOI:10.1002/2211-5463.12994

DR. SARA LIND (Orcid ID : 0000-0002-9510-3816)
PROF. GUNNAR JOHANSSON (Orcid ID : 0000-0002-0553-8331)
Received Date : 09-Mar-2020
Revised Date : 25-May-2020
Accepted Date : 30-Sep-2020
Article type
: Research Article
Inhibition properties of free and conjugated leupeptin analogues
Erika Billingera, d, Johan Viljanenb, Sara Bergström Lindc, e & Gunnar Johanssona*
Department of Chemistry – BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden. E-
mail: gunnar.johansson@kemi.uu.se.
* Corresponding author
a Dept. of Chemistry-BMC, Biochemistry, Uppsala University, BMC, Husargatan 3, Box 576, SE-75123 Uppsala, Sweden
b Dept. of Chemistry-BMC, Organic chemistry, Uppsala University.
c Dept. of Chemistry-BMC, Analytical Chemistry, Uppsala University
.d Bjerking AB Uppsala, Sweden
e
Office For Science and Technology, Uppsala university,
Running title: Conjugation of leupeptin analogues
Abbreviations: Ahx; 6-aminohexanoic acid, MS:mass spectrometry, SPPS: solid phase peptide
synthesis, APTES: aminopropyltriethoxysilane, CDI: carbonyldiimidazole
Keywords: Leupeptin analogs;Conjugation;Inhibition;
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differences between this version and the Version of Record. Please cite this article as doi:
10.1002/2211-5463.12994
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which permits use, distribution and reproduction in any medium, provided the original work is
properly cited.

Tight binding;Inhibitor design
Enzymes: Trypsin IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1
Conflict of interest: None
ABSTRACT
Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following
its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The
specificity of leupeptin is most likely due to the Leu-Leu-Argininal sequence, and its C-terminal aldehyde
group has been suggested to enhance the binding efficiency and to be essential for function. The terminal
aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether
the inhibitory function of leupeptin towards serine proteases is retained after oxidation or reduction of the
aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior
to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced
inhibitory properties as compared to the oxidized form.
Based on these results, new synthetic leupeptin analogues, Ahx-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-
Arg-COOH, were prepared by solid-phase peptide synthesis using the Fmoc strategy. In these analogues,
the N-terminal capping acetyl group was replaced with a 6-aminohexanoyl group to allow conjugation. The
structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry (MS).
Determination of the inhibitory properties against trypsin revealed that these further modified
tripeptides were tight binding inhibitors to their target enzyme, similar to the natural occurring
leupeptin, with Ki values generally in the micromolar range. The Ahx-Phe-Leu-Arg-COOH
analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads.
All conjugates exhibited inhibitory activity in the same range as for the free
peptides.
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INTRODUCTION
Leupeptin (figure 1) is known to be a natural occurring efficient, tight binding inhibitor to various
proteases, in particular serine proteases.1-8 Leupeptin is produced by various species of actinomycetes and
was also confirmed in a number of other families. The production is therefore not species-specific.9 Since
the discovery of leupeptin, several additional peptidyl argininals have been subject to interest due to the
strong and specific inhibition of serine proteases.10 The specificity of leupeptin is most likely due to the
Leu-Leu-Argininal sequence and the C-terminus aldehyde function is suggested to enhance the binding
efficiency and to be essential for the function.1,11,12 It is also likely that the terminal aldehyde group will
make the structure less vulnerable to carboxypeptidases. Aoyagi reported2 that the reduction or oxidation of
the aldehyde group to alcohol or carboxylate, respectively, strongly impaired the inhibition of plasmin and
papain, to the extent that no exact numerical data were given. Our aim here was to investigate whether the
inhibitory function towards serine proteases is retained also after oxidation or reduction of the aldehyde
group. The biosynthesis of leupeptin does indeed occur via a synthesis of the peptide with a C-terminal
carboxyl group, followed by a reduction to aldehyde catalyzed by the enzyme Leupeptin acid reductase
with NADH as electron donor and thermodynamically supported by ATP-hydrolysis.13 In analogy with that
the chemical synthesis of leupeptin is thus more complicated than standard peptide synthesis due to its L-
argininal moiety. Many analogues to leupeptin have nevertheless been synthesized giving a broad view of
structure-function relations for the inhibition of proteases.12,14,15
Here we are using solid-phase peptide synthesis (SPPS) to synthesize two new leupeptin analogues with an
-COOH at the C-terminus instead of an aldehyde, similar to the biosynthetic precursor. Staying at the
precursor stage does thus allow a straightforward solid-phase peptide synthesis. One option to synthesize
leupeptin to its natural state could actually be based on an enzymatic reduction after the solid-phase peptide
synthesis of the oxidized form. As of today, no enzyme that catalyzes the reduction of the C-terminal
carboxyl group of the precursor to aldehyde in Streptomyces is commercially available.13 The N-terminal
acetyl group was in these tripeptide analogues of leupeptin replaced with 6-aminohexanoyl group that
allows an easy path to conjugation while retaining inhibitory activity. The nucleophilic character of amino
groups is frequently used in the conjugation of peptides, amino acids and proteins and a large number of
activations of conjugation carriers have been developed.14-16 The gel support employed here was the bromo
activated Workbeads® matrix, where the conjugation results in a secondary amine. For conjugation to
inorganic oxide phases, an initial silanisation is often used to introduce suitable functional groups. In this
case we have chosen to modify the oxide surfaces with APTES (aminopropyltriethoxysilane), followed by
activation of the amino groups by CDI (carbonyldiimidazole). The final result will be a coupling of the
peptides by a substituted urea structure. Since the peptides only carry a single primary amino group, the
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conjugation is in all cases expected to be uniform and, furthermore, leaving the important arginine
accessible.
RESULTS & DISCUSSION
Oxidation and reduction of leupeptin
The molecular weight of oxidized and reduced state of leupeptin was determined by MS analysis and can
be seen in figure 2 where the oxidized state shows a peak at 443 m/z and the reduced state shows a peak at
429 m/z with z=1 for both peptides. This is in expected accordance with the peak at 427 m/z for natural
leupeptin (table 1). Full m/z-spectra can be found in supporting information figure S1a and S1b.
Inhibitor binding mode
The original natural leupeptin interacts very strongly, but reversibly with trypsin. The backbone of
leupeptin forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a
water molecule. The aldehyde carbonyl of leupeptin is shown to form a hemiacetal bond with the side chain
oxygen of Ser195 in the active site, in which the hemiacetal oxygen atom is pointing out of the oxyanion
hole and forms a hydrogen bond with His57.17 Radinsky18 modeled both orientations of the hemiacetal and
found that the one where the oxygen atom is facing the oxyanion hole had 15% occupancy whereas the one
where oxygen faces the active site His57 had 85% occupancy.
The weaker inhibition by both the oxidized and reduced state of leupeptin can be explained by the loss of
the hemiacetal bond possibility in the active site to Ser 195. Tentative binding modes for original and
modified leupeptin are shown in figure 3. The stronger inhibition by the oxidized form compared to the
reduced form can be explained by the interaction of the negative oxygen in the oxyanion hole, but without
the hemiacetal formation. The reduced state of leupeptin does also lack the possibility to form a hemiacetal
but can still interact through a hydrogen bond to His57. This could be the reason for the pronounced
difference between an apparent Ki of 2.69 μM for the oxidized form and 270 μM for the reduced form
(table 1). Based on these data, we decided to synthesize analogues with terminal carboxyl group.
Synthesis of Ahx-Phe-Leu-COOH & Ahx-Leu-Leu-COOH
The synthetic pathway of a serine protease inhibitor of leupeptin type is a challenge due to the argininal
residue at the C-terminus of their sequence combined with a low yield as an outcome.19 The standard
arginine peptide, on the contrary, requires only straightforward peptide synthesis and is still a powerful
inhibitor. It should also be noted that the biosynthesis proceeds via synthesis of the peptide, followed by a
specific enzyme mediated reduction of the terminal carboxylate group to aldehyde.13 Conclusively,
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chemical synthesis of a precursor peptide combined with a final enzymatic reduction could actually be the
preferred strategy for production of leupeptin analogues with a terminal aldehyde group.
Purification and characterization of Ahx-Phe-Leu-Arg-COOH & Ahx-Leu-Leu-Arg-COOH
The two peptides, Ahx-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-Arg-COOH, were purified with preparative
HPLC. The yields of the peptides were 36% and 74% respectively, based on the capacity of the resin. The
m/z relation were 514 with z = 1 for Ahx-Leu-Leu-Arg-COOH and 548.4 with z = 1 for Ahx-Phe-Leu-Arg-
COOH by LC-MS, which confirmed the molecular masses of the peptides to 513 Da for Ahx-Leu-Leu-
Arg-COOH and to 547 Da for Ahx-Phe-Leu-Arg-COOH.
Kinetic measurements of Ahx-Phe-Leu-Arg-COOH & Ahx-Leu-Leu-Arg-COOH
The apparent Ki value of Ahx-Leu-Leu-Arg-COOH (9.48 μM) and Ahx-Phe-Leu-Arg-COOH (3.42 μM) lie
in the same micromolar range as oxidized leupeptin (table 1 & 3), confirming that the replacement of the
N-terminal capping acetyl group with AHX only has a minor influence. The replacement of one leucine by
phenylalanine does, furthermore, lower the Ki value by a factor 3. This is probably due to the fact that
phenylalanine is more hydrophobic than leucine and interacts more strongly in the active site of the
enzyme. Both the synthesized peptides act as a tight binding inhibitor as can be seen in figure 4. Even
though the hemiacetal function is lost by synthesizing them with a carboxylic acid instead of the functional
aldehyde, these peptides still show significant inhibition properties.
Conjugated Ahx-Phe-Leu-Arg-COOH
Conjugation of Ahx-Phe-Leu-Arg-COOH
Ahx-Phe-Leu-Arg-COOH was conjugated to three different carriers where the conjugated amount was
determined by subtractive absorbance measurements and can be seen in table 2.
Kinetic measurements of conjugated Ahx-Phe-Leu-Arg-COOH
Since the synthetic analogues display the same inhibition mechanisms as the natural leupeptin (tight
binding) the apparent Ki values (table 3) were determined by equation 1 and 2. The corresponding graphs
can be seen below (figure 5). For details regarding TiO2-peptide kinetics, see supporting information figure
S2, table S1.
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The increase in apparent Ki can be ascribed to sterical inaccessibility due to uneven surface in all three
carriers. The peptides can also be conjugated close to each other making the space for several enzyme
molecules less accessible. Regarding the gel beads the peptides can be hidden inside the beads which will
exclude a number of peptides that are conjugated to those positions and thereby not accessible for the
enzymes. The crosslinking that was observed for conjugated protein inhibitors20 is not possible for this
peptide since it only has one easily reacting group for the conjugation to take place which, furthermore, is
placed opposite to the functional group that is crucial for the inhibition. If the peptide is conjugated in a
way that is not sterically inaccessible the inhibition will not be affected by the conjugation. The
calculations regarding the efficiency are based on the free Ahx-Phe-Leu-Arg-COOH as a reference, since
that is the peptide that is conjugated to all three carriers. The efficiency is easiest regarded as the fraction of
inhibitor molecules that are functionally active, rather than a change of intrinsic molecular properties20 and
the loss of efficiency can primarily be ascribed sterical inaccessibility. The efficiency is thus calculated
from the apparent Ki observed as
. The conjugation of the peptide is performed not to
increase the stability but to merely provide a carrier to the small peptide. This may serve both to remove the
inhibitor in a controlled way from a technical sample but also to minimize the risk of the peptide crossing
for example the skin barrier if used in protective formulas. As can be seen, in regard to the efficiency, the
conjugation of Ahx-Phe-Leu-Arg-COOH to ZnO gives an efficiency of 0.77 which is close to the reference
value of 1. The conjugation itself does not affect the peptides inhibition properties. The lower efficiency of
the peptide conjugated to agarose gel beads could be explained by the steric inaccessibility for the enzyme,
due to the possibility of the peptide hidden inside the gel beads. According to the efficiency about 10% of
the conjugated peptides are available for the enzyme.
Inhibition activity analysis of free and conjugate peptide in gelatin layer
The gelatin erosion method that simulates a surface with a protective cover layer is here used to evaluate
the function of the peptide in its different conjugation states where the relative area increase rate is a plotted
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against the increasing inhibitor concentration in figure 6. The curves obtained as a function of
concentration for the free peptide and the TiO2-conjugated peptide, respectively, are virtually identical.
The data obtained for the peptide-ZnO conjugate, on the other hand, result in a notably steeper slope at
lower inhibitor concentration, suggesting a stronger binding. This may tentatively be explained by a model
where the enzyme molecules, once “captured” by the immobilized peptides, also interact nonspecifically
with the ZnO-surface, resulting in addition of binding energies and thus also cooperative binding. As has
been noted earlier by us21 the well area converges to an asymptotic nonzero value “plateau”, at high
inhibitor concentration that is virtually identical for the different cases. The limit may actually be set by the
size of the droplet applied and initial enzyme diffusion. However, the similar limiting values further
confirm that the inhibition modes are identical for free and conjugated inhibitor. The control experiments
carried out with non-conjugated oxide particles resulted in plateau values close to unity, compared to the
value of 0.42-0.50 for the inhibitor formulations (table 4). The plateau value of ZnO-particles decreases,
however, to 0.86 suggesting a certain inhibitory effect by the oxide itself. This is actually in accordance
with the pattern observed for the ZnO conjugate. Taken together, the patterns, for both slope and plateau,
confirm that the conjugation does not influence the peptide function. For details regarding the gelatin
erosion method, see supporting information figure S3, table S2.
CONCLUSIONS
After background experiments where the aldehyde group of leupeptin was converted to carboxylate or
alcohol group, respectively, we could design two new leupeptin analogue protease inhibitors by the use of
solid-phase peptide synthesis. This design of peptides, using SPPS, is useful in order to create smaller
peptides with inhibitory effect in a straightforward and easy synthesis. The two new peptides had a new
functional group, -COOH, instead of the original aldehyde group of the natural state leupeptin. The N-
terminal acetyl group was also replaced by the commonly used conjugation spacer Ahx as an extra handle
in the other end of the peptide for an easier conjugation path. With these modifications the peptides gave an
apparent Ki value in the micromolar range. The conjugation of Ahx-Phe-Leu-Arg-COOH to inorganic
particles and agarose gel beads gave a retained apparent Ki value in the micromolar range for all
conjugates. The conclusions from these data are, first, that the leupeptin analogs still are functional
inhibitors, with a more retained function for the oxidized form. Furthermore, the replacement of the
original N-terminal acetyl group with Ahx did not have a large impact on the inhibitory properties but
allowed well defined and sterically favorable conjugation of the peptide. The data further show that the
properties are retained when the peptide is immobilized to a soluble or particulate carrier. It is possible that
the peptide efficiency may be further improved by application of the natural enzyme aided maturation
reaction of natural leupeptin to generate the aldehyde function. As a consequence, and in a more general
context,, peptide inhibitors that are designed for conjugation may facilitate the use of their inhibitory
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properties both in medical formulations and as enzyme scavengers in biotechnical applications. In the latter
case it allows easy removal of conjugated inhibitors from the sample by filtering or centrifugation. The
highly developed technology for peptide synthesis does also allow both rational and virtually unlimited
combinatorial approach far beyond canonical amino acids for the design of inhibitors.
MATERIALS & METHODS
Materials
Leupeptin (L2884), AgNO3, sodium borohydride, Nα-Benzoyl-DL-arginine-4-nitroanilide hydrochloride
(BAPA), trypsin from porcine pancreas type IX-S, titanium(IV) oxide, zinc oxide, 1,1´-
carbonyldiimidazole (CDI), triethylamine, (3-aminopropyl)triethoxysilane (APTES), dimethylsulfoxide
(DMSO), Acetonitrile (ACN), formic acid (FA), acetic acid, O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-
tetramethyluronium-hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA), dichloromethane
(DCM), piperidine, acetic anhydride, trifluoroacetic acid (TFA), triisopropylsilane (TIS), Fmoc-Leu-OH,
Fmoc-Phe-OH and Boc-Ahx-OH were all purchased from Sigma-Aldrich. WorkBeads™ 40/1000 ACT
was a kind gift from Bio-Works Sweden AB, Uppsala.
Methods
Oxidation of leupeptin using Tollen’s test and confirmation of reaction with mass spectrometry (MS)
0.25 gram of AgNO3 was dissolved in 15 mL milliQ H2O. 0.5 mL 1M NaOH was added creating a brown
solution, which precipitated. The addition of 0.5 mL of 25% NH3 ( 14M) resulted in a clear solution
+
containing Ag(NH3)2 . The function of the solution was confirmed by reaction with benzaldehyde. 100 L
+
of 5.3 mM leupeptin was mixed with 0.5 mL Ag(NH3)2 incubated in water bath (70) for 5 min. The
oxidized inhibitor (figure 7) was desalted by chromatography on ISOLUTE SPE C18. Peptides (625 nM)
were dissolved in 50% ACN 0.1% FA and directly infused by a syringe pump (Harvard apparatur,
Holliston, MA, USA) at a flow rate of 4 L/min. Infused peptides were ionized by electrospray using an
Ion Max Source (ThermoFisher Scientific, Bremen, Germany). Peptide mass spectra were recorded over a
period of 5 min, resulting in >570 spectra per peptide with a LTQ Orbitrap Velos Pro (ThermoFisher
Scientific) in the m/z 150-2000 range using the Orbitrap (Fourier transform) analyzer set to 30 000
resolution. The ionization potential was +4.2 kV, no sheath gas flow was used and the inlet capillary
temperature was set to 300° C. Instrument calibration was carried out according to standard operating
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procedures using Pierce LTQ Velos ESI Positive Ion Calibration Solution (Thermo Fisher Scientific) to
assure high mass accuracy.
Reduction of leupeptin and confirmation of reaction with MS
A 100-fold molar excess of NaBH4 was added to a solution of 0.53 M leupeptin dissolved in milliQ-H2O.
The reduction (figure 8) took place over a time period of 2 hours, followed by desalting with SPE C-18 and
was confirmed with MS-detection using the same protocol as in “Oxidation of leupeptin”.
Synthesis of Ahx-Phe-Leu-Arg-COOH & Ahx-Leu-Leu-Arg-COOH
Solid-phase peptide synthesis & purification
The peptides were synthesized using Fmoc chemistry on a 50 mol scale, starting from Fmoc-Arg(Pbf)
Wang resin (70 mg, 0.7 mmol/g). Deprotection of the Fmoc groups was carried out using 20% piperidine in
dimethyl formamide (DMF). Amino acid couplings were performed in DMF using amino
acid:HCTU:DIPEA (5:4:10), with coupling times of 30 min to 1h. Peptides were quantified using Kaisers
test.22 Total deprotection and simultaneous cleavage from the solid support was achieved with a mix of
TFA/TIS/H2O (95/2.5/2.5%, v/v, 5 mL) for 1.5 h with agitation. After filtration and evaporation of TFA by
N2(g) bubbling, the peptides were precipitated and washed once with cold diethyl ether (Et2O), followed by
lyophilization. For details see supporting information.
The crude peptide was purified with reversed phase HPLC (Varian 940-LC) using a semi preparative Grace
Vydac C8-column (22 mm x 150 mm, 10 µm, 300 Å) with a gradient of ACN (acetonitrile):H2O,
containing 0.1% TFA, from 5% (2 min) to 40% ACN in 20 minutes at a flow rate of 15 mL/min. UV
detection was done at 220 nm. The peptide identities were confirmed by LC-MS using an instrument
constellation consisting of a Waters 2700 sample manager, an Agilent 1100 series HPLC/UV-VIS diode-
array detector, and Waters Micromass ZQ mass detector in negative ion mode. The measured m/z values
(546.3 and 512.3, respectively) were in accordance with the calculated masses (figure 4). Fractions
containing the desired peptides (figure 9) were pooled, evaporated, freeze-dried and stored at -20 C until
further use.
Conjugation of Ahx-Phe-Leu-COOH
Inorganic particle carriers
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All derivatization and immobilization steps were performed at room temperature and in plastic Falcon
tubes or 2 mL plastic Eppendorf tubes. 0.5 grams of the inorganic particulate carriers (TiO2 & ZnO) was
first silanized using 100 mM APTES in 10 mL ACN while stirring for 24 hours. The particles were then
centrifuged followed by removal of ACN, washed 3 times with EtOH, one final time with acetone and then
dried at 65 C for 5 hours. Following that, the particles were activated using 120 mg CDI and 0.72 mmol
triethylamine in 5 mL ACN while stirring for 2 hours. The particles were then centrifuged followed by
removal of ACN, washed 3 times with EtOH, one final time with acetone and then dried at 65 C o/n.
Conjugation of Ahx-Phe-Leu-Arg-COOH was carried out by stirring a controlled amount of peptide
overnight with 50 mg activated TiO2 or ZnO in 0.1M NH4HCO3 buffer pH 7.52. The particles were then
centrifuged, and the absorbance of the supernatants was measured at 259 nm, allowing a subtractive
quantification of conjugated peptide using the extinction coefficient of 195 M-1cm-1 for Phe residues23. The
conjugation was terminated by washing with 0.1M NH4HCO3 pH 7.52 and the conjugated particles (figure
10) were stored at 4 C until further use.
Agarose gel beads
WorkBeads™ 40/1000 ACT from Bio-Works with an average particle size of 45 µm and reactive group
content of 200 µmol/mL were used when coupling Ahx-Phe-Leu-Arg-COOH to agarose gel beads (resin).
1 mL resin was washed with deionized water on a glass filter and dried using suction. 0.5 mL 4.38 mM of
peptide 1 in 0.1M NH4HCO3 pH 7.52 was then added to 0.5 mL dried resin. The slurry mixture was
incubated at RT overnight while stirring. Following that, the slurry mixture was centrifuged, and
absorbance was measured on the supernatant. The amount conjugated was determined as mentioned above
in ‘Inorganic particle carriers’. The immobilization was terminated by first washing the resin with 0.1M
NH4HCO3 pH 7.52 and drying by suction followed by incubation of the resin in 1M ethanolamine-HCl pH
9.5 at RT overnight while stirring to block the remaining reactive groups. The blocking agent was finally
removed by washing the gel beads as described above. The suspension of the conjugate (figure 11) was
stored at 4C in 0.1M NH4HCO3 pH 7.52 until further use.
Kinetics
Real time measurements
Kinetic parameters were obtained by measuring initial velocities in the presence of selected concentrations
of natural leupeptin (0.14-1.62 µM), oxidized leupeptin (8-127 µM), reduced leupeptin (10-413 µM) and
peptide 1 & 2 (8-254 µM). All reactions were performed in 0.1 M NH4HCO3 pH 7.52 and 2% (v/v) DMSO
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using concentrations of BAPA and trypsin fixed at 1 mM and 0.25 µM, respectively. The reaction was
monitored at 410 nm in a Shimadzu UV-1601 UV-vis spectrophotometer.
End point measurements
Kinetic parameters were obtained by mixing substrate, enzyme and conjugate in a plastic Eppendorf tube
with varying concentrations of TiO2-peptide 1 0-70 M, ZnO-peptide 1 0-60 M and gel beads conjugated
to peptide 1 0-60 M and incubating with end-over-end rotation. 150 µL aliquots where withdrawn at 5
time points for each reaction and the reaction was stopped in 100 mM acetic acid pH 3. Absorbance was
measured at 410 nm in a UV-1601 UV-vis spectrophotometer (Shimadzu).
For real time and end point measurements the Ki value was estimated by the graphical method of Dixon24
and Morrison25 (equation 1). Ki was extracted after fitting the data to equation 1 and equation 2 using
GraphPad Prism 8. A KM of 0.82 mM was used for the calculations.
(Eq. 1)
The form of Kiapp varies with the type of inhibitor. For competitive inhibitors, as in the case of leupeptin,
the Ki value can be calculated from equation number 2:
(Eq. 2)
Inhibition activity analysis of free and conjugate peptide in gelatin layer
The experiments were performed as described earlier.21 The concentration of the free inhibitor Ahx-Phe-
Leu-Arg-COOH was ranging from 5 to 50 μM, for ZnO-conjugate 1-15 μM and for TiO2-conjugate 1-25
μM.
AUTHOR CONTRIBUTION
GJ designed the project, served as senior and corresponding author, academic supervisor for EB.
EB shared the design planning, wrote the first draft of the manuscript, performed or participated in most
experiment and participated in the final writing steps.
JV provided expertise in designing and leading the peptide synthesis work and participated in the writing of
the manuscript.
SL planned and performed MS experiments and participated in the writing of the manuscript.
ACKNOWLEDGEMENT
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We want to thank Professor Thomas Norberg for sharing his vast knowledge in peptide synthesis. The
work was financially supported by Bo Rydins foundation for scientific research, Magnus Bergvall
Foundation (SBL) and Swedish Foundation for Strategic Research (SBL).
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Figure Legends
Figure 1. Leupeptin in its natural state (Ac-Leu-Leu-Arg-CHO)
Figure 2. (a) MS spectra of oxidized leupeptin with a peak at 443 m/z corresponding to the molecular ion
[M+H], (b) MS spectra of reduced leupeptin with a peak at 429 m/z corresponding to the molecular ion
[M+H] confirming the identity of both products.
Figure 3. Tentative comparison of active site binding mode for original leupeptin, reduced and oxidized
leupeptin, respectively.
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Ltd.

Figure 4. The ratio vi/v0 is plotted as a function of the inhibitor peptide Ahx-Phe-Leu-Arg-COOH to the
left and Ahx-Leu-Leu-Arg-COOH to the right. The conditions for the experiments and calculations were
[Trypsin] = 0.25 μM, [BAPA] = 1 mM, Km = 0.82 mM. Error bars representent SD
Figure 5. vi/v0 is plotted as a function of the conjugated TiO2-Ahx-Phe-Leu-Arg-COOH (a), ZnO-Ahx-Phe-
Leu-Arg-COOH (b) and WorkBeads™-Ahx-Phe-Leu-Arg-COOH (c). The conditions for the experiments
and calculations were [Trypsin] = 0.25 μM, [BAPA] = 1 mM, Km = 0.82 mM. Error bars representent SD
Figure 6. Relative area increase rate for the free peptide 1, conjugated peptide 1 and free particles.
Figure 7. The oxidation of leupeptin using Tollen’s Test.
Figure 8. The reduction of leupeptin using sodium borohydride.
Figure 9. Structure of the two tripeptides.
Figure 10. Conjugate of Ahx-Phe-Leu-Arg-COOH to TiO2 particles ,
where R = (CH2)3NHC(=O)-Ahx-Phe-Leu-Arg-COOH.
Figure 11. Conjugate of agarose gel bead and peptide 1. The gel bead in comparison with the peptide is
about 40 000 times larger and the gel bead can conjugate a number of peptides. This figure only
demonstrates the conjugation between the bead and the peptide itself and the components are not in correct
scale.
Supporting information
Supplement titles
Figure S1a. MS spectra of oxidized leupeptin.
Figure S1b. MS spectra of reduced leupeptin.
Figure S2. Kinetic measurements of TiO2-peptide conjugate.
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Ltd.

Figure S3. Erosion well formation rate for the free Ahx-Phe-Leu-Arg-COOH and in its conjugated
state.
Table S1. Extracted values from figure S2.
Table S2. The rates for the gel experiment for free Ahx-Phe-Leu-Arg-COOH and its conjugated
state to TiO2 and ZnO.
Data accessibility
For information about raw data, i.e. direct instrumental output, contact
gunnar.johansson@kemi.uu.se
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Ltd.

Tables
Table 1. Summary of the representative results from the reduction and oxidation of leupeptin,
including the obtained kinetic parameters for the different state of the inhibitors.
State
Functional group
Ki
m/z observed
z
Mw (Da)
88 ± 8
nM
Natural
-CHO
427.3
+1
426.3
2.7 ± 0.1
Leupeptin
Oxidized
Reduced
-COOH
-CH2OH
443.3
429.3
+1
+1
442.3
428.3
μM
270 ±
100 μM
Table 2. Summarizes the amount of Ahx-Phe-Leu-Arg-COOH immobilized onto the chosen carriers.
Conjugated amount of
Alternative
Carriers
Ahx-Phe-Leu-Arg-COOH
TiO2
ZnO
6.1 nmol/mg particle
4.4 nmol/mg particle
150 nmol/ml beads
Inorganic particles
Gel beads
WorkBeads™
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Table 3. Summary of the apparent Ki values from the different states of inhibitors and the efficiency
where free Ahx-Phe-Leu-Arg-COOH is used as the reference. *reference value
State
Carrier
Peptide
Leupeptin
Ki
Efficiency
Ki stored
88 ± 8 nM
2.7 ± 0.1 μM
-
-
Leupeptin-COOH
Free
-
Ahx-Leu-Leu-Arg-COOH
Ahx-Phe-Leu- Arg-COOH
9.5 ± 1.0 μM
3.4 ± 0.2 μM
11 ± 2 μM
-
1.00*
0.32
0.77
0.12
TiO2
ZnO
7.1 ± 1.1 μM
7.6 ± 1.5 μM
53 ± 9 μM
Conjugated
Ahx-Phe-Leu- Arg-COOH
4.4 ± 1.3 μM
29 ± 4 μM
WorkBeads™
Table 4. The relative asymptotic plateau values extracted from GraphPad Prism 8, where the
resulting value shows the asymptotic area ratio obtained at virtually maximal inhibition.
State of peptide 1
Plateau
Free Ahx-Phe-Leu-Arg-COOH
Only ZnO-particles
0.50  0.04
0.86  0.07
0.46  0.02
1.24  0.04
0.42  0.21
ZnO-Ahx-Phe-Leu-Arg-COOH
Only TiO2-particles
TiO2-Ahx-Phe-Leu-Arg-COOH
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feb4_12994_f1.pdf
Figure 1. Leupeptin in its natural state (Ac-Leu-Leu-Arg-
CHO)
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feb4_12994_f2.pdf
b.
a.
443.298
429.318
100
100
90
80
70
60
50
40
30
20
90
80
70
60
50
40
30
20
10
0
443.335
430.322
444.301
10
0
444.338
444.446
443.219
443.2
429.241
429.2
442.558
442.6
442.771
442.8
443.075
443.0
443.579 443.763
443.6 443.8
m/z
444.077 444.223
444.0 444.2
444.704
444.6 444.8
444.913
445.0
428.892
429.091
429.442 429.543 429.673
429.4 429.6
429.824 429.925
429.8 430.0
430.095
430.244
430.2
430.390
430.4
430.576
430.6
443.4
444.4
428.8
429.0
m/z
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feb4_12994_f3.pdf
SER
OH
H
HIS
H
CH2OH
SER
HIS
H
N
NH2
O
C
O
O
H
H
H
N
OH
NH
O-
CH3
N
N
H
H
O
Reduction
SER
H
N
NH2
O
O
OH
H
N
H
NH
HIS
CH3
N
N
H
O
H
H
O
H
C
O
O
H
Oxidation
Original leupeptin
H
N
NH2
O
O
H
N
OH
NH
CH3
N
N
H
H
O
O
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feb4_12994_f4.pdf
b.
a.
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feb4_12994_f5.pdf
a.
b.
c.
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feb4_12994_f6.pdf
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feb4_12994_f7.pdf
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feb4_12994_f8.pdf
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feb4_12994_f9.pdf
Ahx-Phe-Leu-Arg-COOH
547.7 Da
Ahx-Leu-Leu-Arg-COOH
513.7 Da
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feb4_12994_f10.pdf
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feb4_12994_f11.pdf
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