A caged substrate peptide for matrix metalloproteinases

Article abstract of DOI:10.1039/c4pp00297k

Based on the widely applied fluorogenic peptide FS-6 (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂; Mca = methoxycoumarin-4-acetyl; Dpa = N-3-(2,4-dinitrophenyl)l-α,β-diaminopropionyl) a caged substrate peptide Ac-Lys-Pro-Leu-Gly-Lys-Lys-Ala-Arg-NH<

Full text of DOI:10.1039/c4pp00297k

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Photobiological Sciences
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PAPER
A caged substrate peptide for matrix
metalloproteinases
a,b
Cite this: DOI: 10.1039/c4pp00297k
c,e
a
a
Elena Decaneto,* Stefania Abbruzzetti, Inge Heise, Wolfgang Lubitz,
d,e
a,b
Cristiano Viappiani* and Markus Knipp
Based on the widely applied fluorogenic peptide FS-6 (Mca–Lys–Pro–Leu–Gly–Leu–Dpa–Ala–Arg–NH
Mca = methoxycoumarin-4-acetyl; Dpa = N-3-(2,4-dinitrophenyl)l-α,β-diaminopropionyl) a caged sub-
strate peptide Ac–Lys–Pro–Leu–Gly–Lys*–Lys–Ala–Arg–NH (*, position of the cage group) for matrix
2
;
2
metalloproteinases was synthesized and characterized. The synthesis implies the modification of a carba-
midated lysine side-chain amine with a photocleavable 2-nitrobenzyl group. Mass spectrometry upon UV
irradiation demonstrated the complete photolytic cleavage of the protecting group. Time-resolved laser-
flash photolysis at 355 nm in combination with transient absorption spectroscopy determined the bi-
a b
phasic decomposition with τ = 171 ± 3 ms (79%) and τ = 2.9 ± 0.2 ms (21%) at pH 6.0 of the photo
Received 1st August 2014,
Accepted 8th November 2014
induced release of the 2-nitrobenzyl group. The recombinantly expressed catalytic domain of human
membrane type I matrix metalloproteinase (MT1-MMP or MMP-14) was used to determine the hydrolysis
efficiency of the caged peptide before and after photolysis. It turned out that the cage group sufficiently
shields the peptide from peptidase activity, which can be thus controlled by UV light.
DOI: 10.1039/c4pp00297k
www.rsc.org/pps
substrates and are, therefore, involved in processes like wound
healing, bone resorption, embryonic development, tissue
Introduction
7
–10
The use of caged compounds for the light triggered release of development etc.
Consequently, diseases like arthritis,
biologically active compounds has been demonstrated in many rheumatism, and cancer are often linked to MMP dysfunc-
1
–3
6,8
cases.
The development of fully optical control for effector tion. Even if MMPs share the zinc binding motif conserved
activation has proven to be useful in a number of applications sequence HEXGHXXGXXH and show essentially identical
where in situ modulation of the substrate concentration with three dimensional structure, their substrate specificity is very
3
–5
11
spatial and temporal resolution is important.
In specific different. Therefore, the design of pharmaceutically appli-
cases, fast decomposition of photoactivated compounds has cable MMP inhibitors requires detailed knowledge about sub-
allowed the temporal limitations imposed by rapid mixing strate–enzyme interactions and their catalytic process.
6
techniques to be overcome. However, the application of light
Recently, the study of the role of water dynamics in the sub-
triggered activation of the large group of hydrolytic enzymes strate–enzyme interaction of the membrane type I matrix
called peptidases is a yet underrepresented issue. An impor- metalloproteinase (MMP14 or MT1-MMP) has revealed that a
tant class of peptidases is comprised by the zinc-dependent strong coupling between water and protein motion is crucial
1
0,12,13
matrix metalloproteinases (MMPs), of which more than 20 for substrate recognition.
MT1-MMP has been reported
different isoforms are expressed in humans. They play a major to cleave several types of collagen, e.g. type I, II, III collagens,
role in the degradation and remodeling of extracellular matrix and to hydrolyze a variety of different substrates such as
κ-elastin, gelatin, β-casein, and other extracellular matrix com-
1
4
ponents. The active site contains a catalytic zinc atom bound
to three histidines and exposed to the solvent. The structure of
the substrate binding site is characterized by a pocket located
next to the zinc atom called “S1 pocket”, which is probably a
determining factor of substrate specificity. This hydrophobic
pocket has different depths among MMP isoforms and dictates
a
Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470
Mülheim an der Ruhr, Germany. E-mail: elena.decaneto@cec.mpg.de;
Tel: +49-208-306-3684
b
Department of Physical Chemistry II, Ruhr University Bochum, Universitätstrasse
1
c
50, D-44780 Bochum, Germany
Department of Life Sciences, University of Parma, Parco Area delle Scienze 11A,
I-43124 Parma, Italy
15
the substrate docking. The suggested catalytic mechanism of
d
Department of Physics and Earth Sciences, University of Parma, Parco Area delle
action for MMPs involves the nucleophilic attack of water
Scienze 7A, I-43124 Parma, Italy. E-mail: cristiano.viappiani@fis.unipr.it;
Fax: +39-0521-905223; Tel: +39-0521-905208
2
+
molecules polarized by the catalytic Zn and the conserved
glutamate on the scissile bond at close-to-neutral pH. For this
e
NEST, Istituto Nanoscienze, CNR, Piazza San Silvestro 12, 56127 Pisa, Italy
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to happen, a substrate must be bound and form a Michaelis Peptide synthesis
1
6
complex. Even if several specificity studies using collagen-
The peptides Fluo-LK, Fluo-KK, KK, and Ø-KK presented in
Scheme 2 were synthesized by standard solid phase synthesis
1
1
model peptides have been published, the specific mechan-
ism of MMP–substrate interaction and recognition remains an
important unresolved issue. Here we report on the design and
synthesis of a MT1-MMP caged substrate peptide. The pro-
ducts of light decomposition are characterized by mass spec-
(
SPS; Rink amide ChemMatrix, Aldrich) using Fmoc chemistry
and N-methylpyrrolidone as the solvent. In the case of the
caged peptide Ø-KK the coupling efficiency of the modified Lys
was 84.7%, as estimated from UV absorption of the fulvene
trometry.
Laser-flash
photolysis
experiments
allow
δ
adduct formed upon removal of the N -Fmoc protecting group
determining an upper limit for substrate release by light. The
applicability of the photolabile caged MT1-MMP is demon-
strated by fluorescence and HPLC detection.
with 25% piperidine/DMF for 20 min at room temperature. For
the synthesis of Ø-KK and the fluorogenic peptides FS-1 and
FS-6, care was taken to minimize exposure to light. All peptides
were purified by C -RPC (Phenomenex Gemini C18, 110 Å,
1
8
5
µm, 250 mm × 21.2 mm) using HPLC and a linear gradient
of CH CN from 0 to 90% in 0.1% trifluoroacetic acid (TFA).
Peptides were characterized by ESI MS: Fluo-LK: m/z Found:
3
Materials and methods
Materials
The expression plasmid for the catalytic domain of human
MT1-MMP (residues 112–292) was transformed into Escherichia
coli BL21(DE3) (Novagen). Expression and purification of the
1
7
enzyme were carried out as described previously.
Synthesis of nitrobenzyl-L-lysine
Synthesis of 2-nitrobenzylcarbonyl chloride (3) (3.13 g, light
brown crystals). 1.68 g (11 mmol) of 2-nitrobenzylalcohol (1)
and 4.36 g (22 mmol) of diphosgene (2) were dissolved in THF
and refluxed for 2 h. The solvent was removed under reduced
pressure at 50 °C. mp 59 °C (from 3 : 2 diethyl ether–pentane);
+
m/z 169 (M , 17%), 152 (2), 136 (44), 125 (37), 121 (11),
2
1
3
04 (32), 99 (12), 91 (72), 78 (100), 63 (74), 51 (49), 44 (26), Scheme 1 Synthetic route for the preparation of N -(9-fluoromethoxy-
6
carbonyl)-N -2-nitrobenzyloxycarbonyl-L-lysine as
a precursor for
H 2 2 3 4
9 (17), 30 (5); NMR: δ (500 MHz; CD Cl ; (CH ) Si) 5.73 (2 H,
4
5
peptide synthesis.
s, CH ), 7.59 (1 H, m, Ph-C H), 7.66 (1 H, m, Ph-C H),
7
CDCl
2
6
3
C
.75 (1 H, d, Ph-C H), 8.18 (1 H, d, Ph-C H); δ13 (400 MHz;
3
4
3
; CDCl
3
) 67.8 (CH
2
), 125.4 (Ph-C ), 128.8 (Ph-C ), 129.6
6
1
6
(
Ph-C ), 130.1 (Ph-C ), 134.2 (Ph-C ), 147.2 (C–NO ), 147.5
2
(
COCl).
2
6
Synthesis of N -(9-fluoromethoxycarbonyl)-N -2-nitrobenzyl-
oxycarbonyl-L-lysine (5) (2.8 g, 15.5% light yellow crystals). All
steps were carried out in the dark. 12.33 g (33.5 mmol) of
2
N -(9-fluoromethoxycarbonyl)-L-lysine
(4)
(Fmoc–Lys–OH)
(Bachem) was suspended in 200 mL of DMF and incubated
with 1.1 g (33.5 mmol) of 3 and 4.63 g (33.5 mmol) of K CO .
2
3
The mixture was stirred for 6 h at 50 °C. Afterwards 600 mL of
CH Cl were added and extracted with saturated NaHCO solu-
2
2
3
tion and twice with saturated NaCl solution. Upon filtration
the solution was dried over MgSO and the solvent removed
under reduced pressure. The brown liquid was dissolved in
5 : 5 CH Cl –CH OH and purified by silica gel chromato-
graphy. Elution was achieved with 85 : 15 CH Cl –CH OH.
NMR: δH (400 MHz; d -DMSO; (CH )Si ) 8.08 (1 H, d, C H–
4
9
2
2
3
2
2
3
3
6
3
4
4
/5
9
CNO
(
2
), 7.86 (2 H, d, Fmoc-C H), 7.77 (1 H, t, Fmoc-C H), 7.30
2
/7
1/3/6/8
2 H, m, Fmoc-C H), 7.40 (4 H, m, Fmoc-C
H), 7.6 (3 H,
H), 5.33 (3 H, s, NH), 4.2 (3 H, m, Fmoc-CH /Lys-
4
/5/6
m, Ph-C
2
Scheme 2 Substrate peptides for MT1-MMP referred to in this study.
Mca methoxycoumarin-4-acetyl; Dnp 2,4-dinitrophenyl;
-nitrobenzyloxycarbonyl; Dpa = N-3-(2,4-dinitrophenyl)-L-α,β-diami-
α
C ), 3.32 (broad, OH), 3.16 (2 H, s, CH
), 1.6 (2 H, m, Lys-C H
.26 (2 H, m, Lys-C H ).
2
–C–CNO
2
), 3.95 (2 H,
=
=
Ø =
ε
β
δ
m, Lys-C H
1
2
2
), 1.38 (2 H, m, Lys-C H ),
2
2
γ
nopropionyl. ∼ denotes the hydrolyzed peptide bond.
2
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+
1
263.7 [M + H] . Calc.: 1263.6; Fluo-KK: m/z Found: 1278.6, Design of a photocaged MMP substrate
Calc.: 1278.7; KK: m/z Found: 938.6, Calc.: 939.6; Ø-KK: m/z
Found: 1117.7, Calc.: 1117.6; For Ø-KK: λmax(water)/nm 266
23
According to the nomenclature of Schechter and Berger, the
main subsite for substrate recognition in MMPs is the speci-
ficity pocket S1′. As the structure of S1′ varies between MMP
3
−1
−1
(
ε/dm mol cm 3914).
24
family members, modification of the P1′ side-chain allows the
Enzyme kinetics of fluorescent peptide substrates
25–27
modification of substrate specificity.
Thus, it was our
Enzyme kinetic parameters were determined by monitoring aim to inhibit substrate cleavage by a sterically demanding
the increase in fluorescence intensity of Mca due to the modification of P1′ using a photocleavable group so that
1
8
decrease of FRET quenching by Dnp upon hydrolysis of the photolytic release provides the substrate peptide.
fluorogenic peptides Fluo-KK and Fluo-LK with a 96-well plate
reader connected to a fluorescence spectrophotometer (Varian tion kinetics is the fluorogenic peptide FS-6 (see Scheme 2)
A widely used substrate peptide for the study of MMP reac-
2
0
Cary Eclipse) at 25 °C. Fluorescence at λem = 400 nm was moni- with the non-natural amino acid N-3-(2,4-dinitrophenyl)-
tored upon excitation at λex = 340 nm. To measure the rate of L-α,β-diaminopropionyl (Dpa) as the quencher group in position
substrate cleavage, an enzyme was added to the reaction P2′. It was previously demonstrated that substitution of Dpa by
6
mixture in 50 mM Tris/HCl (pH 7.4), 100 mM NaCl, 5 mM N -2,4-dinitrophenyl-L-lysine (Lys(Dnp)) results in a substrate for
1
0
CaCl2 and varying concentrations of fluorescent substrates MT1-MMP termed Fluo-LK (Scheme 2). We determined the
2–15 µM). The kinetic data were fitted by a pseudo-first-order enzyme kinetic constants for Fluo-KK applying Michaelis–
(
reaction rate (eqn (1)) using OriginLab 8.6 (Microcal)
Menten kinetics (Fig. 1). As a result, both the secondary rate
constant kcat/KM as well as the Michaelis constant KM are com-
parable to the values obtained for FS-6 (Table 1), indicating that
substitution of Dpa by Lys(Dnp) is well tolerable.
FðtÞ ¼ Fmax½1 ꢀ eꢀ ꢁ þ F0
kt
ð1Þ
where F(t) is the fluorescence at time t, F and F
are the
max
0
For the modification of the P1′ side-chain, the insertion of
a chemical functionality is mandatory. Because a structure of
MT1-MMP with a substrate peptide or product peptides is cur-
initial and the final fluorescence intensities, respectively, and
1
9
k is the pseudo-first-rate constant k = [E]tot (kcat/K
M
). The con-
centration of MT1-MMP and fluorescent substrates was deter-
1
7
−
1
−1
rently not available, a superposition of its inhibitor bound
mined by UV-VIS absorption (ε275 nm = 35 410 M cm and
2
8
1
−1
X-ray structure (PDB code 1BQQ) with the X-ray structure of
ε
410 nm = 7500 M− cm for the enzyme and fluorogenic sub-
2
0
MMP12 containing the two product peptides Pro–Gln–Gly and
strates respectively).
2
9
Ile–Ala–Gly in the catalytic site (PDB code 2OXZ) was per-
formed (Fig. 2). It should be noted that MMP12 and MT1-
MMP share a high degree of similarity in the active site archi-
tecture. Fig. 2 suggests that the side-chain of P1′ of the
product peptide Ile–Ala–Gly is directed toward the interior of
S1′. However, S1′ is comprised of a very deep pocket, much
larger than would be required to host the isobutyl side-chain
of P1′, suggesting that a long side-chain with a functional
group like Lys could substitute this position and the peptide
would still be cleaved. To the best of our knowledge, despite
much effort taken towards the design of optimal synthetic sub-
Analysis of the hydrolysis of non-fluorescent peptides
.5 µM MT1-MMP in 50 mM Tris/HCl (pH 7.4), 100 mM NaCl
and 5 mM CaCl was incubated with 1 mM of either KK or
Ø-KK for 2.5 h at 25 °C. Aliquots of 150 µL were taken every
0 min and immediately frozen in liquid nitrogen. Quantifi-
2
2
3
cation of peptides was performed by C18-RPC (Phenomenex
Gemin, C18, 110 Å, 5 µm, 250 mm × 4.6 mm) using HPLC and
absorbance detection at 202 and 265 nm. A linear gradient of
−
1
3
CH CN from 0 to 90% in 0.1% TFA, 1.0 ml min was applied.
3
0
strate for MMPs, Lys in P1′ was usually not considered.
Laser flash photolysis
To test if Lys can be inserted into the site P1′, the peptide
Fluo-KK was synthesized as a derivative of Fluo-LK (Scheme 2).
As can be seen in Fig. 1, Fluo-KK is a reasonable substrate for
MT1-MMP. Determination of the enzyme kinetic constants
demonstrates that its affinity and specificity are approx. one
order of magnitude smaller compared to Fluo-LK (Table 1).
However, the parameters are very similar to another widely
applied MMP substrate peptide FS-1 (see Scheme 2). As a result,
Lys at P1′ can indeed be used and the amine group is available
for modification with a photoremovable protecting group.
The kinetics of Ø-KK photodecomposition were studied by
2
1
means of transient absorption. A solution of Ø-KK in 50 mM
Tris/HCl (pH 7.4), 100 mM NaCl, 5 mM CaCl at 25 °C in a
quartz cuvette was photo excited by the third harmonic (λ =
2
3
–
55 nm) of a nanosecond, Q-switched Nd:YAG laser (Surelite II
10, Continuum, FWHM 5 ns, 10 mJ) which entered the
22
cuvette at 90° to the detection beam. The monitoring beam was
provided by the cw output of a 75 W Xe arc lamp (Oriel) that
was filtered with a 405 nm interference filter. The beam was
then passed through a monochromator (Oriel MS257), and its
intensity was monitored by a 5-stage photomultiplier (Applied
Photophysics) and digitized by a digital oscilloscope (LeCroy
Results and discussion
Synthesis of a caged substrate peptide
LT374). The experiment was repeated with Ø-KK in N saturated
2
water whose pH was adjusted by adding small amounts of con-
centrated NaOH or HCl solutions, and in 10 mM 2-(N-morpho- In comparison with the direct coupling of the 2-nitrobenzyl
lino)ethanesulfonic acid (MES)/NaOH (pH 6.0).
group to a side-chain amine, linkage via a carbamido group
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Fig. 2 Superposition of the X-ray structure of MT1-MMP (PDB code
28
29
1
BQQ) with that of MMP12 (PDB code 2OXZ) with soaked in product
peptides Pro–Gln–Gly (P1 and P2) and Ile–Ala–Gly (P1’ and P2’).
ESI MS demonstrated the integrity of Ø-KK (Fig. 3). More-
over, upon UV irradiation the mass peak corresponded to the
peptide without the cage group (KK, Scheme 2), demonstrating
that photoconversion was indeed obtained.
Mechanism of photolysis
A usable caged compound requires a significantly reduced
inhibitory activity and fast recovery of the activity after cleavage
1
,4,32,33
of the photolabile group by light.
On the basis of pre-
vious experimental evidence on related systems, we propose
that the release mechanism of the photocleavable group is as
Fig. 1 (A) Kinetic parameters kcat and K
lysis of different concentrations of Fluo-LK (blue line) and Fluo-KK (red
line) by MT1-MMP in 50 mM Tris/HCl (pH 7.4), 100 mM NaCl, 5 mM
M
were obtained for the hydro- described by Scheme 3. Experimental evidence for this mecha-
nism is presented and discussed in the following section.
Flash photolysis experiments indicate that the photogen-
erated nitronic acid intermediates 6 of 2-nitrobenzyl com-
pounds undergo deprotonation in water at neutral pH with
CaCl
Hydrolysis of Fluo-LK (blue line) and Fluo-KK (red line) by MT1-MMP.
[E ] = 0.35 µM in 50 mM Tris/HCl (pH 7.4), 100 mM NaCl, 5 mM CaCl
2
2
at 25 °C. Data were fitted by the Michaelis–Menten equation. (B)
(
0
7
8
−1
34–36
at 25 °C, [S] = 10 µM). First-order rate constants, kobs, were obtained by rates in the 10 –10 s range.
fitting eqn (1); second order rate constants kcat/K
The deprotonation rate is
M
were calculated from
essentially determined by the pKa of the particular nitronic
k
obs/[E ] (see Table 1).
0
acid intermediate, with the more acidic compounds having
M M
Table 1 Second-order rate constant kcat/K , Michaelis constant K for
the hydrolysis of Fluo-LK and Fluo-KK in comparison with commercially
available fluorogenic peptides (FS-1 and FS-6, see Scheme 1) for
MT1-MMP at 25 °C
(M− s−1
1
)
Substrate
pH
kcat/K
M
M
K (µM)
2
2
0
0
5
FS-1
FS-6
7.5
7.5
7.4
6
3.9 × 106
n.d.
7.9
36 ± 15
40 ± 18
5.7 ± 1.3
1.3 × 10
5
Fluo-KK
Fluo-KK
Fluo-LK
(0.7 ± 0.5) × 10
(0.11 ± 0.02) × 10
(2.2 ± 0.2) × 10
4
6
7.4
n.d., not determined.
requires less synthetic effort, and higher yields are obtained.
Such a peptide was previously reported to release an inhibitory
peptide against the calmodulin dependent protein kinase II
upon photoactivation. Scheme 2 shows that the synthesis of
the caged Fmoc-L-lysine 5 requires only two steps of decent
yield and can be easily inserted as a resin in routine SPS as
was demonstrated here in the case of Ø-KK.
Fig. 3 ESI MS of Ø-KK before (green) and after photolysis (black). 1 mg
of Ø-KK was dissolved in 3 mL of 5 mM N-ethylmorpholine/acetic acid
3
1
(
(
pH 7.4). Photocleavage was performed through irradiation at 366 nm
8 W, ≈1.2 mW m ) for 1 h. Ø-KK: m/z: obs. 1117.7, calc. 1117.6; KK:
−2
m/z: obs. 938.8, calc. 939.6. The inset shows the absorption spectrum
of Ø-KK.
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Scheme 3 Reaction mechanism of the photolysis of Ø-KK.
3
7–39
higher dissociation rates.
For instance, the nitronic acid of the nitronic acid, we observe a slower rising kinetics charac-
generated by photolysis of 2-nitrobenzaldehyde has a pK value terized by a lifetime of (96 ± 9) ns. This transient is most likely
a
8
−1
of 2.1 ± 0.1 with a deprotonation rate exceeding 10
s
,
associated with deprotonation of the nitronic acid to give the
whereas for 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) the aci-nitro anion, as suggested by studies on related com-
4
4,45
pK of the corresponding nitronic acid is 3.69 ± 0.05, with a pounds.
Similarly, the rise in the signal measured on
a
7
−1 21
deprotonation rate of (1.58 ± 0.09) × 10 s
.
The decompo- caged sulfate occurs with a lifetime of (64 ± 7) ns, as previously
2
1
sition reaction proceeds from 6 through the bicyclic intermedi- reported. At about 1 µs, equilibrium is reached for the aci-
ate 7. The lifetime of this intermediate can be very different nitro intermediate for both compounds. The aci-nitro inter-
depending on the leaving group and is dramatically increased mediates for Ø-KK and for caged sulfate decay had lifetimes of
2
1,40,41
in the presence of a buffer at neutral pH.
decomposition of 7, the carbamidated peptide 9 is autocon- limit for the release of 9 in well buffered neutral solutions.
verted to the final product KK under protonation of the Lys When the aci-nitro intermediate was monitored in N saturated
nitrogen and the release of CO . The carbamate decarboxyla- water (pH 5.8), decay was found to occur through a biexponen-
Upon (840 ± 20) ms and (61 ± 1) ms, respectively. This sets a lower
2
2
9
6
−1 −1
tion occurs at a rate of 10 to 10 M
s
, which was deter- tial relaxation, with lifetimes (3.9 ± 0.1) ms (31%) and (414 ±
mined for numerous different carbamidates at a pH of 15) ms (69%). In a buffered solution at lower pH (10 mM MES/
4
2
6
.8–13.0. Detailed studies of the caged glutamate N-1-(2- NaOH, pH 6.0) a similar biexponential behavior was obtained
nitrophenyl)ethoxycarbonyl-L-glutamate revealed that the de- (τ = (171 ± 3) ms (79%); τ = (2.9 ± 0.2) ms (21%)). The reason
a
b
4
3
carboxylation is not rate limiting between pH 5 and 8. Fig. 4 for the biexponential relaxation is at present not understood
shows the absorbance changes at 405 nm of a buffered (pH and will require future investigations.
7
.4) aqueous solution of Ø-KK, following a single, 15 mJ laser
Using caged sulfate as an actinometer, and considering
pulse at 355 nm. For comparison, Fig. 4 also shows the signal that the chromophoric properties of Ø-KK and caged sulfate
observed after photolysis of caged sulfate under identical con- are essentially identical, the absorbance changes at 1 µs can
ditions. After a fast, unresolved rising phase due to formation be used to estimate the yield for formation of the aci-nitro
intermediate. Using the yield determined for caged sulfate
(
0.47), we can estimate an upper limit for the yield of the
aci-nitro intermediate of Ø-KK as 0.24 ± 0.05.
Cleavage of non-fluorescent peptides
The qualitative comparison of the hydrolysis rate for the case
of the caged and non-caged substrates is important to demon-
strate the utility of Ø-KK as a caged-compound in experiments
in the presence of an enzyme. This study was done by time
course C18-RPC measurements of the MT1-MMP + Ø-KK + KK
mixture. Simultaneous detection of absorbencies at 202 nm
and 265 nm allowed distinguishing between peptides contain-
ing a 2-nitrobenzyl group and those without. Assignment of
the peaks was performed by chromatographic separation of KK
and Ø-KK. The peaks corresponding to the product peptides
were obtained from the extended incubation of KK and Ø-KK
with MT1-MMP in the dark.
Fig. 4 Time resolved absorbance changes at 405 nm following
exposure to 15 mJ laser shots for caged sulfate (green) and Ø-KK in
Fig. 5A shows the chromatogram of the intact substrates
2
50 mM Tris/HCl (pH 7.41), 100 mM NaCl, 5 mM CaCl (blue) and in
1
0 mM MES/NaOH (pH 6.0) (grey). The cyan curve shows the aci-nitro before addition of the enzyme in which KK and Ø-KK are well
transient absorbance for Ø-KK in nitrogen saturated water (pH 7.0). Red
solid lines are the results of a fit to an exponential rise in the nano-
seconds and a mono- or bi-exponential decay in the long milliseconds.
For the cases of Ø-KK in nitrogen saturated water and in MES buffer it
was not possible to observe the rise of the signal in the nanoseconds,
due to intense scattered light. See the text for fitting parameters.
separated. Fig. 5B and C exhibit the chromatogram of a
sample upon incubation with MT1-MMP for 30 min and 2.5 h,
respectively, at 25 °C. An additional peak is observed after
30 min and corresponds to one the of KK fragments. After 1 h,
a fourth peak appeared with absorption at 265 nm, that has
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characterized. Enzyme kinetic studies demonstrated that the
substrate resin P1′, which is typically Leu can be substituted by
Lys and still be effectively cleaved. The introduction of the
Lys5 amine function allowed the attachment of a photolabile
o-nitrobenzyl group in the substrate peptide Ac–Lys–Pro–Leu–
Gly–Lys–Lys–Ala–Arg–NH
2
.
The new compound Ø-KK
(
Scheme 1) is easy to synthesize and is highly soluble in water.
ESI MS demonstrated that light induced removal of the nitro-
benzyl group was successful and that no significant side reac-
tion had occurred. The kinetics of photodecomposition after
nanosecond laser photolysis has been characterized by time-
resolved transient absorbance spectroscopy. The protolytic dis-
sociation of the aci-nitro compound 6 through the bicyclic
intermediate 7 is pH dependent and occurs with τ = 171 ms
a
in buffered solutions at pH 6.0. At this pH, a second faster
decay (21%) occurs with τ = 2.9 ms (Fig. 4).
b
Analysis of the enzymatic hydrolysis by MT1-MMP using
18-RPC demonstrated that the cleavage of the caged-substrate
C
can be neglected in the presence of the non-caged peptide.
This demonstrates that Ø-KK is sufficiently sterically hindered
to allow incubation and activation by UV light.
To the best of our knowledge, this is the first light-con-
trolled caged peptide suitable for matrix metalloproteinases.
Fig. 5 HPLC profiles upon incubation of MT1-MMP with KK and Ø-KK
(
A) before addition of the enzyme, (B) after 30 min and (C) after 2.5 h.
The peaks were assigned as follows: (1) KK, (2) Ø-KK, (3) KK fragment
Ac–Lys–Pro–Leu–Gly or Leu–Lys–Ala–Arg–NH , (4) Ø-KK fragment
(D): product–substrate ratio versus incu-
Acknowledgements
2
This work was supported by the Cluster of Excellence RESOLV
2
Lys(Ø)–Lys–Ala–Arg–NH .
(
EXC 1069) funded by the Deutsche Forschungsgemeinschaft
bation time.
and the Max Planck Society. We are grateful for the technical
assistance of Heinz-Werner Klein, (MPI for Coal Research,
been attributed to the Lys(Ø)–Lys–Ala–Arg–NH2 fragment of Mülheim, Germany) and Nina Breuer (MPI for Chemical
Ø-KK (Scheme 2). The time course of the peptide cleavage is Energy Conversion, Mülheim, Germany).
shown in Fig. 5 (bottom) demonstrating that Ø-KK cleavage
occurs at a much smaller rate compared to KK (>10-fold) and
can be neglected in the presence of uncaged compounds.
Despite the negligible hydrolysis of the caged-substrate com-
Notes and references
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