Synthesis and kinetic studies of a low-molecular weight organocatalyst for phosphate hydrolysis in water

Article abstract of DOI:10.1039/b914974k

Kinetic studies of a low-molecular weight organocatalyst 1 are presented. Compound 1 contains two histidines and one cationic side chain attached to a central aromatic core. In aqueous solution 1 accelerates the hydrolysis of a prototypal phosphodiester w

Full text of DOI:10.1039/b914974k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and kinetic studies of a low-molecular weight organocatalyst
for phosphate hydrolysis in water†
Michael Merschky and Carsten Schmuck*
Received 23rd July 2009, Accepted 1st September 2009
First published as an Advance Article on the web 30th September 2009
DOI: 10.1039/b914974k
Kinetic studies of a low-molecular weight organocatalyst 1 are presented. Compound 1 contains two
histidines and one cationic side chain attached to a central aromatic core. In aqueous solution 1
accelerates the hydrolysis of a prototypal phosphodiester with rate enhancements of up to two orders of
magnitude. A detailed HPLC analysis of hydrolysis experiments in Bis-Tris-buffer showed that the
buffer itself can act as a nucleophile at least with the cyclic phosphate 16. Compound 1 is also an
efficient host for the binding of bis-(para-nitrophenyl)-phosphate 14 with extraordinary high affinity of
Kass = 24 400 M-1 in buffered water.
attached to a central core providing a minimalistic concave active
Introduction
site. The capability of 1 to enhance phosphodiester cleavage
Enzymes are among the most efficient catalysts known so far
was tested on three prototypal aromatic phosphodiesters. For
hydrolysis of the cyclic catecholphosphate 1610 in aqueous buffer
and can exhibit extraordinarily large rate enhancements and
selectivity. Therefore, there has been a longstanding interest in
solution, efficient catalysis with a rate enhancement of ca. 102 over
developing low molecular weight mimics of natural enzymes.
the background reaction is observed.
However, initial work on enzyme models either often failed or
suffered from serious limitations.1 For example, it has not been
possible so far to design a functional model for the catalytic
triad of chymotrypsine and related enzymes.2 Perhaps, the best
Results and discussion
Design and synthesis of compound 1
and most prominent biomimetic enzyme models reported are
the cyclodextrin based RNAse A mimics developed by Breslow
in the 1990s.3 However, in recent years numerous peptide based
organocatalysts have been developedfor various applications.4 The
focus here is not necessarily on exactly mimicking the structural
features of a specific active site (as in the early attempts on enzyme
mimics) but on using the same functional groups also employed
by enzymes such as imidazoles (in the amino acid histidine), OH
groups (as in serine or threonine), ammonium or guanidinium
cations (lysine and arginine) or carboxylates (aspartate and
glutamate). For example, histidine based peptides have been shown
to exhibit hydrolytic activity.5 A recent development reported by
Baltzer is the phosphorester hydrolysis of RNA mimic substrates
catalyzed by a 42 residue peptide that folds into a helix-loop-helix
motif providing an active site based on histidine and arginine
residues.6 Very recently we have shown that the incorporation
of a non natural amino acid with tailor made properties into
this peptide can even further enhance the catalytic activity of the
system.7 We could also show that histidine containing octapeptides
hydrolyze phosphates and esters in aqueous buffer solution.8 In
other work, Reymond reported that peptide fragments such as
His-Ser incorporated into a dendritic polymer have esterase-like
activity.9 In this context we report here the first examination
of phosphodiester hydrolysis induced by compound 1, which
contains two histidines and an additional cationic side chain
The design of molecule 1 was based on the following ideas: The
two histidine residues should function as acid–base-catalysts and
the guanidiniocarbonyl-pyrrole cation could either serve as a
cationic binding site, which should allow substrate complexation
and/or electrostatic stabilization of the transition state or provide
additional nucleophilic catalysis due to the pKa of ca. 7 of this
residue.11 Octapeptides with this combination of amino acids had
been found to be efficient catalysts for phosphate ester hydrolysis
as identified from the screening of a split-mix library with
625 members in previous work.8 Of course, the same pattern of
functional groups is also found in natural enzymes such as RNAse
A. Its active site contains two histidines (His 12 and 119) and one
lysine (Lys 41). The lysine binds the negatively charged phosphate
and electrostatically stabilizes the transition state and the two
histidines perform a bifunctional acid–base-catalysis.12 We now
wanted to test whether this combination of residues is also active
outside a classical peptide environment. We therefore decided
to attach these three groups to an aromatic triamine13 as the
central core. The alternative arrangement of the three functional
side chains and the three ethyl groups directs the functional side
chains onto the same side of the aromatic ring, creating a concave
arrangement hopefully capable to bind and hydrolyze phosphor
esters.
The synthesis of 1 is shown in Scheme 1. Triamine 2 was first
converted in a one-pot reaction to the di-Boc-mono-Cbz-protected
template 3 by slowly adding both protecting reagents (Cbz-Cl
and Boc2O) simultaneously to a solution of 2 in water/dioxane.
This way, the orthogonally protected 3 was obtained in 21%
isolated yield. After removal of the Cbz-group (H2/Pd), the
Institute for Organic Chemistry, University of Duisburg-Essen, Univer-
sita¨tsstraße 7, 45141 Essen, Germany. E-mail: carsten.schmuck@uni-due.de;
Fax: (+49) 201 183 4259; Tel: (+49) 201 183 3097
† Electronic supplementary information (ESI) available: 1H NMR spectra
of 1 and 16. See DOI: 10.1039/b914974k
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guanidinocarbonyl-pyrrole moiety was coupled to the amino
group of 4 in form of the di-Cbz-protected amino acid 5 using
PyBOP as coupling reagent. Deprotection of the Boc-protecting
groups in 6 with TFA gave the diamine 7. Acetylated histidine
was then attached to yield the protected precursor 8, using
the same coupling conditions as before. Finally, the remaining
Cbz-protecting groups in 8 were cleaved off (H2/Pd) to provide
compound 1.
Scheme
2
Synthesis of the di-Cbz-protected artificial arginine
analogue 5.
Binding studies with bis-(para-nitrophenyl)-phosphate (14)
The first substrate tested to evaluate the hydrolytic efficiency of 1
was the bis-(para-nitrophenyl)-phosphate 14, because of its easily
UV/Vis-spectrophotometrically detectable leaving group. Hence,
14 (5 mM) was mixed with 10 mol-% 1 (0.5 mM) in aqueous
Bis-Tris-buffer (20 mM) at pH 6. This pH was chosen to ensure
partial protonation of both histidines so that they could function
both as acid and base (or nucleophile, respectively). Unexpectedly,
precipitation occurred on mixing of the reactants. Even though
the precipitate could be dissolved by the addition of 5% DMSO
to the reaction mixture, no hydrolysis of 14 could be detected
by UV and HPLC analysis. As the formation of a precipitate
indicated formation of a complex between 14 and 1, we checked
this by means of fluorescence titration. Addition of aliquots of a
stock solution of 14 (400 mM in 2 mM Bis-Tris-buffer at pH 6
with 5% DMSO) to a solution of 1 (20 mM in the same buffer
mixture) indeed leads to a significant decrease of the fluorescence
at 330 nm (Fig. 1 top). A non-linear curve fit of the binding
isotherm (Fig. 1 bottom) taking dilution ◦into account provides
a binding constant of K = 24 400 M-1 at 23 C. Hence, 14 is bound
surprisingly strong by 1 taking into account that this complex
formation takes place in water. This strong complexation most
likely also prevents hydrolysis of the substrate when 14 is bound
in an orientation which would not allow subsequent hydrolysis
by the two histidines. The calculated energy minimized structure
of the complex as obtained after a Monte Carlo conformational
search (Macromodel V 8.0, OPLS 2005, water solvation model,
1000 steps) is shown in Fig. 2. According to the molecular
mechanics calculation the anionic phosphate is bound by the
guanidiniocarbonyl-pyrrole cation with additional assistance of
one histidinium cation. The second histidine p-stacks with one of
the para-nitrophenyl rings.
Scheme 1 Synthesis of compound 1.
The di-Cbz-protected artificial arginine analogue 5 needed for
the synthesis of 1 was synthesized starting from pyrrole aldehyde
914 (Scheme 2). The guanidino group was introduced by reacting
the free carboxylic acid group in 9 with Cbz-protected-guanidine
(= Cbz-Gua) using PyBOP in DMF as the coupling reagent to
give 10. After potassium permanganate oxidation of aldehyde 10
to the carboxylic acid 11, N-Cbz-protected 1215 was coupled to
the pyrrole-carboxylic acid to give the methyl ester 13, which was
hydrolyzed with lithium hydroxide in a mixture of THF/H2O to
provide the di-Cbz-protected artificial arginine analogue 5.
Hydrolysis of 2-hydroxypropyl-4-nitrophenyl-phosphate (15)
We next selected phosphate 15 as substrate which is often used as
a model substrate in phosphor ester hydrolysis studies. We also
hoped that it would show a lower binding strength to 1 compared
to 14, due to the missing second para-nitrophenyl moiety. Indeed,
in the presence of 1 substrate 15 is hydrolyzed faster than without.
However, the kinetic data could not be analyzed straightforwardly
because the reaction did not follow either a clear first or a
second order rate profile. Furthermore, the rate enhancement with
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Fig. 3 Concentration time profile for the hydrolysis of 15 (1 mM) in the
presence of varying amounts of 1 (Bis-Tris-buffer pH 6, 20 mM).
Hydrolysis of catechol-phosphate 16 in Bis-Tris-buffer
We then turned to catechol phosphate 16 as a substrate, which was
also used by Breslow in his work on cyclodextrin based RNAse
A mimics.3 In Bis-Tris-buffer (pH 6.1, 20 mM) hydrolysis of 16
(5 mM) induced by 1 (0.5 mM) at 20 ◦C was indeed observed
(Fig. 4). The disappearance of the starting material was followed
by HPLC showing that within 20 hours about 50% of 16 are
hydrolyzed in the presence of 1 compared to less than 20% in its
absence.
Fig. 1 Fluorescence binding study. Addition of equivalents of 14 (400 mM
stock solution) to a solution of 1 (20 mM) in Bis-Tris-buffer (pH 6.1,
23 ◦C) leads to a significant decrease of the fluorescence (top). Analysis
of the binding isotherm (bottom) provides a binding constant of K =
24 400 M-1.
Fig. 2 Energy minimized structure of the complex between 1 and
phosphodiester 14 (Macromodel V8.0, OPLS 2005, water solvation
model).
50 mol-% 1 is already the same as with 100 mol-% (Fig. 3). This
might indicate the formation of a 2:1 complex between 15 and 1
under these conditions. With respect to the strong complexation
of bis-(para-nitrophenyl)-phosphate 14 by 1 this appears to be
plausible. We therefore did not pursue the kinetic analysis of
the hydrolysis of 15 any further. Especially, as additional studies
further showed (vide infra) that the use of Bis-Tris-buffer is not
without problems.
Fig. 4 Hydrolysis of 16 (5 mM) with (᭹) and without (ꢀ) added 1
(0.5 mM = 10 mol-%) in aqueous Bis-Tris buffer (pH 6.1, 20 mM).
However, HPLC analysis (Fig. 5) revealed that under these con-
ditions in the uncatalyzed and catalyzed reaction 16 (rt, 4.2 min)
is not only hydrolyzed to the open catechol monophosphate
17 (rt 3.1 min) but also two additional hydrolysis products are
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Hydrolysis of catechol-phosphate 16 in phosphate-buffer
As Bis-Tris-buffer cannot be used as a buffer, we switched to
phosphate-buffer as an alternative. Of course, the phosphate in
the buffer is a competitive binding substrate itself for the cationic
catalyst 1. Therefore, to achieve catalyzed hydrolysis in phosphate-
buffer is much more challenging than in Bis-Tris-buffer due to the
weaker binding strength of the substrate. First, we optimized the
reaction conditions. The concentration of the phosphate buffer
directly affects the rate of the background reaction. After 24 hours
about half of the substrate 16 (initial concentration 5 mM) is
hydrolyzed in 30 mM phosphate buffer of pH 6.3 (16/17 =
57:43) whereas in 150 mM buffer nearly 90% had already reacted
(16/17 = 87:13). This increased background rate, however, is not
simply due to the increased ionic strength of the buffer as the
addition of the equivalent amount of NaCl did not affect the
reaction rate. Most likely, phosphate can act also as nucleophile
and opens the catechol phosphate to form a diphosphate which
is then hydrolyzed by water. We found the best compromise
for a slow background reaction but sufficient buffer capacity
to assure pH constancy during the reaction is to apply 1 mM
substrate 16 in 20 mM phosphate-buffer supplemented with 10%
DMSO for solubility reasons. These conditions were then used
throughout the following kinetic analyses. The reaction was again
followed via HPLC with benzamide as an internal standard. A
representative chromatogram is shown in Fig. 6. In the presence
of 1, the hydrolysis of 16 is significantly faster than the background
reaction. Quantitative analysis of the disappearance of 16 showed
that the hydrolysis clearly is a pseudo-first order process. A plot of
log (c/c0) versus time gives a straight line (Fig. 7). Therefore, even
in the presence of a 20-fold excess of phosphate anions, 1 acts as
a catalyst for phosphodiester hydrolysis in water. Furthermore, 1
does not suffer from product inhibition, as complete conversion
takes place even at 10 or 30 mol-% of 1
Fig. 5 HPLC analysis of the hydrolysis reaction of 16 in the presence of
1 as function of time (1 = 0 h, 2 = 20 h, 3 = 44 h, 4 = 70 h, 5 = 140 h).
Compound 1 elutes much later (not shown). (RP 18 Supelcosil 25 cm,
4.6 mm, 5 mm; aqueous Bis-Tris-buffer pH 6.1, 20 mM).
formed (broad peaks at rt 8.8 and 9.5 min, respectively). In the
uncatalyzed reaction, these two products are formed in a ratio of
ca. 1:1 and in similar amounts to the monophosphate 17. In the
catalyzed reaction, 17 is formed in significantly larger amounts
but also the ratio of the two side products changes to ca. 2:1. LC-
MS analysis revealed that these two unexpected peaks are buffer-
adducts (both M = 381.119 g mol-1), and therefore hydrolysis
products in which one of the OH groups of the Bis-Tris-buffer
acted as the nucleophile. As the Bis-Tris-buffer contains two dif-
ferent types of OH groups, the formation of two different products,
statistically in 3:2 ratio if the hydroxyl groups had the same nucle-
ophilicity, are expected (Scheme 3). Bis-Tris-buffer is therefore not
suitable as a buffer system to follow the hydrolysis of 16 quanti-
tatively as 1 obviously increases both the reaction with water and
with the buffer (indicated by the changed ratio of the two buffer
adducts relative to the background reaction). Thus a word of cau-
tion seems appropriate: A simple UV kinetic study would not have
revealed the problem of the buffer reacting as a nucleophile. Any
attempted quantitative analysis and interpretation without this in-
formation, however, would be rather meaningless as it would have
been based on the wrong reaction scheme. The observed transes-
terfication with Bis-Tris-buffer in this context could be a problem
with most organic buffer systems carrying hydroxyl groups.
Fig. 6 Representative HPLC chromatogram of the hydrolysis of 16 in the
presence of 1 in phosphate buffer at different times (1 = 0 h, 2 = 20 h, 3 =
40 h, 4 = 50 h). (RP 8 Supelcosil 25 cm, 4.6 mm, 5 mm; 15% MeOH and
85% aqueous phosphate-buffer pH 6.1, 10 mM).
The catalytic rate enhancement (kcat) increases with increasing
concentration of 1. Unfortunately, in the accessible concentration
range it was not possible to unambiguously determine saturation
kinetics since the solubility of 1 is limited to the lower millimolar
Scheme 3 Bis-Tris-buffer reacts as a nucleophile, opening phosphate 16
to form two isomeric adducts (M = 381.119 g mol-1).
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Fig. 8 Hydrolysis of 16 in imidazole-buffer (pH 6.1, 20 mM) with
100 mol-% 1 (᭹) and without (ꢀ) added 1.
Fig. 7 Normalized concentration-time plot of the hydrolysis of 16 (1 mM)
in the presence of varying amounts of 1 c(1) = 0.0 mM (ꢀ), 0.3 mM (᭹),
2 mM (ꢀ) and 5 mM (᭢) in water (20 mM phosphate buffer, pH 6.1, 10%
DMSO).
range in water. The available data are thus limited to the initial part
of any possible saturation profile. Assuming a classical Michaelis-
Menten type behaviour an extrapolation of the concentration de-
pendence of the overall rate (kobs) on the ratio c(1)/c(16) indicates
a maximum rate within the complex of kcat= 1.4 ¥ 10-2 min-1 and
KM = 123 mM. Hence, according to this extrapolation phosphodi-
ester 16 is hydrolyzed by 1 280 times faster than in the uncatalyzed
reaction (kuncat = 5.0 ¥ 10-5 min-1). However, substrate binding
under these challenging conditions (20 mM phosphate-buffer) is
only weak (Kass = 8 M-1, a reasonable value for substrate binding
under these conditions), so that the overall catalytic efficiency as
expressed as kcat/KM achieves only 0.1 M min-1.
Fig. 9 pH-Dependence of the rate of hydrolysis of 16 (1 mM) catalyzed
by 1 (2 mM) in aqueous phosphate-buffer (pH 6.1, 20 mM, 10% DMSO).
Further elucidation of the mechanism of catalysis
To check that the increased reaction rate in the presence of 1 is
not simply due to nucleophilic or general base catalysis by the two
histidines in 1, we also measured the hydrolysis of 16 in 20 mM
imidazole-buffer. As expected, the rate of the uncatalyzed reaction
is about 3-fold increased relative to the reaction in phosphate-
buffer (kuncat = 1.5 ¥ 10-4 min-1 versus kuncat = 5.0 ¥ 10-5 min-1,
respectively). Imidazole is a much better nucleophile than water
or phosphate, thereby speeding up the background reaction.
Nevertheless, the addition of 1 again leads to a significant increase
in the reaction rate (Fig. 8). This proves that 1 does not simply act
by base or nucleophilic catalysis as imidazole alone does.
1 (measured pKa values are 6.0 and 6.9). This might be a hint for
a bifunctional acid–base catalysis as is also found in the natural
enzyme RNAse A17 (which shows a pH optimum around 6–7) as
well as in Breslow’s cyclodextrin RNAse A mimics.
To exclude that metal contamination is responsible for the
enhanced hydrolysis of 16, we repeated the kinetic runs in the
presence of 1 equivalent of EDTA relative to 1. As evident from
Fig. 10, the reaction rate does not change either for the background
reaction or for the catalyzed reaction. Therefore, metal ions are
not involved in the hydrolysis.
In phosphate-buffer the rate of the reaction in the presence of
1 is pH-dependant with an optimum around pH 6.1–6.2 as seen
from the pH profile in Fig. 9. The uncatalyzed reaction is pH
dependant as well, with a linear increase from pH ca. 6.2 onwards
most likely due to the general base or nucleophilic catalysis by the
phosphate-buffer. It should be mentioned here, that the binding
strength of the guanidiniocarbonyl-pyrrole binding motif is pH
sensitive as well. Therefore, a straightforward analysis of this pH-
profile with respect to the kinetic data is not possible. Nevertheless,
since the kcat data are corrected for the uncatalyzed reaction, the
pH profile cannot be due to simple medium effects16 but must be
caused by either changes in the binding affinity or the catalysis
itself. The maximum of the pH dependence of kcat at pH 6.1–6.2
is within the region of pKa-values of the two histidine moieties in
Conclusions
We present here a new small molecular weight compound 1
which induces the hydrolysis of the phosphodiester model system
16 under the challenging conditions of an aqueous phosphate
buffer. In contrast to natural enzymes with their hydrophobic
microenvironment around the active center, shielding the catalysis
process, it is very difficult to design small molecules without such
a hydrophobic shielding that are capable of enhancing phosphor
ester hydrolysis in water.18 In the case of phosphodiester 14 we
achieved a very high binding constant in water, but this inhibited
hydrolysis, showing the complexity of finding a well balanced
compromise between binding strength and chemical reactivity.
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53% yield). mp: 143-145 ◦C; Rf: 0.50 (SiO2, dichloromethane/ethyl
acetate = 7/3); 1H-NMR (400 MHz, [d6]-DMSO) d = 5.18 (s, 2H,
benzyl-CH2), 6.95-6.99 (m, 2H, pyrrole-CH), 7.34-7.42 (m, 5H,
aryl-CH), 8.75 (br.s, 1H, gua-NH), 9.44 (br.s, 1H, gua-NH), 9.68
(s, 1H, aldehyde-CH), 11.11 (br.s, 1H, gua-NH), 12.41 (br.s, 1H,
pyrrole-NH);13C-NMR (100 MHz, [d6]-DMSO) d = 66.5 (benzyl-
CH2), 114.7, 117.1 (pyrrole-CH), 127.8, 128.0, 128.4 (aryl-CH),
134.8 (pyrrole-Cq), 136.2 (aryl-Cq), 158.6, 181.3 (CO and CN).
-1
˜
FT-IR n (KBr-pellet) [cm ] = 3392 [m], 3273 [m], 1733 [m], 1671
[s], 1643 [s], 1532 [m], 1339 [m], 1277 [m], 1217 [s]; HR-MS (pos.
ESI) m/z = 337.091 0.005 (calculated for C15H14N4O4 + Na+:
337.091).
5-(N-(Benzyloxycarbonyl)carbamimidoylcarbamoyl)-1H-pyrrole-
2-carboxylic acid 11
Fig. 10 Catalyzed and uncatalyzed hydrolysis of 16 with (circles) and
without (triangles) added EDTA analyzed as a first order kinetic.
To a solution of the pyrrole aldehyde 10 (320 mg, 1.02 mmol) in
acetone (5 ml) a suspension of potassium permanganate (322 mg,
2.04 mmol) in acetone/water (1/1; 20 ml) was added dropwise over
a period of 1 h. The solution was heated for 1 h at 40 ◦C and stirred
at room temperature for an additional hour. Sodium dithionite
(17.7 mg, 0.10 mmol) was added and the reaction mixture was
filtered through a bed of celite and thoroughly washed with
aqueous sodium hydroxide solution (30 ml, 5%). The combined
filtrates were acidified with hydrochloric acid (5%). The resulting
colorless precipitate was filtered off, washed with hydrochloric
acid (5%) and dried over phosphorus pentoxide to get the pyrrole
carboxylic acid 11 (307 mg, 0.93 mmol, 91% yield) as a colorless
powder. mp: >250 ◦C; Rf: 0.4 (SiO2, dichloromethane/methanol
= 9/1); 1H-NMR (400 MHz, [d6]-DMSO) d = 5.27 (s, 2H, benzyl-
Beside this we could show the importance for method development
in such hydrolysis experiments. The observed side reactions with
organic buffer systems like Bis-Tris would lead to incorrect results
if a simple UV detection were used instead of the HPLC detection
method used here. Finally, the hydrolysis of cyclic phosphate 16 is
accelerated in the presence of 1 by up to a factor of 102 proving that
even this minimalistic model compound 1 is capable of hydrolysing
phosphor esters in water. However, substrate binding under these
conditions is still rather weak limiting the overall activity of 1.
Experimental section
General information
3
CH2), 6.82 (d, J = 4.0 Hz, 1H, pyrrole-CH), 7.37-7.44 (m, 6H,
Reaction solvents were dried and distilled under nitrogen before
use. All other reagents were used as obtained from Aldrich, Merck,
Lancaster, Acros or Fluka. The 1H NMR spectra were recorded at
250 MHz or 400 MHz and the 13C NMR spectra were recorded
at 62.5 MHz or 100 MHz at room temperature, using d6-DMSO
or CDCl3 as solvent. The chemical shifts are reported relative to
the solvent signals. The apparent coupling constants are given
in Hz. The description of the fine structure is as follows: s =
singlet, br.s = broad singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br = broad signal. Peak assignments are based on
DEPT, 2D NMR studies and/or comparison with literature data.
All IR spectra were measured as KBr-pellets on a PerkinElmer FT-
IR 1600 spectrophotometer. The maxima are classified in three
intensities: s (strong), m (medium), w (weak) and are reported
in cm-1. Melting points were measured in open end glass capillary
tubes and are uncorrected.
aryl-CH and pyrrole-CH), 9.46 (br.s, 1H, gua-NH), 9.97 (br.s, 1H,
gua-NH), 12.57 (br.s, 1H, pyrrole-NH); 13C-NMR (100 MHz, [d6]-
DMSO) d = 68.0 (benzyl-CH2), 115.3, 116.5 (pyrrole-CH), 127.3
(pyrrole-Cq) 128.3, 128.5, 128.6 (aryl-CH), 129.7 (pyrrole-Cq),
˜
135.0 (aryl-Cq), 153.4, 154.7, 160.6, 161.0 (CO and CN); FT-IR n
(KBr-pellet) [cm-1] = 3355 [m], 3330 [s], 3238 [w], 2980 [m], 2589
[w], 1755 [m], 1697 [s], 1643 [s], 1591 [s], 1351 [w], 1288 [w], 1233
[s], 1179 [s], 764 [s]; HR-MS (neg. ESI) m/z = 329.089 0.005
-
(calculated for C15H13N4O5 : 329.089).
(S)-Methyl 3-(5-(N-(benzyloxycarbonyl)carbamimidoylcar-
bamoyl)-1H-pyrrole-2-carboxamido)-2-(benzyloxycarbonyl-
amino)-propanoate 13
The pyrrole carboxylic acid 11 (270 mg, 0.82 mmol) and PyBOP
(425 mg, 0.82 mmol) were dissolved in DMF (10 ml) and NMM
(1.5 ml). The methyl ester 12 (260 mg, 0.90 mmol) was added and
the yellow solution stirred overnight at room temperature. Cold
water (50 ml) was added and the precipitate was filtered through
a sintered glass funnel. The filtrate was extracted with ether (3 ¥
20 ml) and the combined organic fractions were washed twice with
brine (20 ml). The solvent was removed in vacuo and the resulting
orange solid was purified together with the filtered precipitate via
flash column chromatography (SiO2, ethyl acetate/cyclohexane
7/3). The coupling product 13 (290 mg, 0.◦51 mmol, 63% yield) was
isolated as a pale orange solid. mp: 101 C; Rf: 0.50 (SiO2, ethyl
N-(N-(Benzyloxycarbonyl)carbamimidoyl)-5-formyl-1H-pyrrole-
2-carboxamide 10
A mixture of the pyrrole carboxylic acid 9 (291 mg, 2.10 mmol),
PyBOP (1.64 g, 3.14 mmol) and NMM (1 ml) in dry DMF
(10 ml) was stirred for 10 min at room temperature. Cbz-guanidine
(607 mg, 3.14 mmol) was added and the resulting solution stirred
overnight. The solution was hydrolyzed with water (30 ml) and the
precipitate was filtered through a Buchner funnel. The filter cake
was washed with water and ether. The crude product was purified
via column chromatography (SiO2, ethyl acetate/dichloromethane
3/7) to get the light red coupling product 10 (350 mg, 1.11 mmol,
1
acetate/cyclohexane = 7/3); H-NMR (400 MHz, [d6]-DMSO)
d = 3.52-3.59 (m, 2H, CH2), 3.62 (s, 3H, CH3), 4.27-4.32 (m,
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3
1H, CH), 5.04 (d, J = 2.8 Hz, 2H, benzyl-CH2), 5.14 (s, 2H,
7/3); 1H-NMR (400 MHz, [d6]-DMSO) d = 1.05 (t, 3J = 7.2, 9H,
CH2-CH3), 1.38 (s, 18H, Boc-CH3), 2.65-2.68 (m, 6H, CH2-CH3),
4.18 (d, 3J = 4.6, 4H, CH2), 4.25 (d, 3J = 4.4 Hz, 2H, CH2), 5.04
(s, 2H, benzyl-CH2), 6.51 (s, 2H, NH), 7.14 (s, 1H, NH), 7.34
(br.s, 5H, aryl-CH); 13C-NMR (100 MHz, CDCl3) d = 16.6 (CH2-
CH3), 23.1 (CH2-CH3), 28.6 (Boc-CH3), 38.9, 39.4 (CH2-NH),
67.0 (benzyl-CH2), 79.7 (Boc-Cq), 128.2, 128.3, 128.6 (aryl-CH),
benzyl-CH2), 6.78 (d, 3J = 3.4 Hz, 1H, pyrrole-CH), 6.96 (s, 1H,
pyrrole-CH), 7.30-7.39 (m, 10H, aryl-CH), 7.73 (d, 3J = 8.0 Hz,
1H, NH), 8.49 (s, 1H, NH), 8.77 (br.s, 1H, gua-NH), 9.36 (br.s, 1H,
gua-NH), 11.12 (br.s, 1H, gua-NH), 11.85 (br.s, 1H, pyrrole-NH);
13C-NMR (100 MHz, [d6]-DMSO) d = 52.1 (CH3), 53.7 (CH),
65.7, 66.2 (benzyl-CH2), 112.0 (pyrrole-CH), 127.2 (pyrrole-Cq),
127.6, 127.7, 127.8, 128.3, 128.4, 128.7 (aryl-CH), 136.7 (aryl-Cq),
132.2, 132.8 (Cq-CH2-NH), 136.6 (aryl-Cq), 143.9, 144.1 (Cq-
-1
-1
˜
˜
155.9, 160.0, 171.0 (CO and CN); FT-IR n (KBr-pellet) [cm ] =
CH2-CH3), 155.5, 155.9 (CO); FT-IR n (KBr-pellet) [cm ] = 3343
3385 [s], 2953 [w], 1735 [s], 1636 [s], 1551 [s], 1465 [m], 1282 [s],
1213 [s], 1048 [w], 752 [w], 697 [w]; HR-MS (pos. ESI) m/z =
587.186 0.005 (calculated for C27H28N6O8 + Na+: 587.186).
[s], 2975 [s], 2932 [m], 2874 [w], 1685 [s], 1519 [s], 1391 [w], 1365
[m], 1247 [s], 1167 [s], 1045 [m], 867 [w], 773 [w], 699 [s]; HR-MS
(pos. ESI) m/z = 606.351 0.005 (calculated for C33H49N3O6 +
Na+: 606.351).
(S)-3-(5-(N-(Benzyloxycarbonyl)carbamimidoylcarbamoyl)-1H-
pyrrole-2-carboxamido)-2-(benzyloxycarbonylamino)propanoic
acid 5
tert-Butyl (5-(aminomethyl)-2,4,6-triethyl-1,3-phenylene)bis-
(methylene)dicarbamate 4
A mixture of the pyrrole methyl ester 13 (270 mg, 0.48 mmol)
and lithium hydroxide (22.9 mg, 0.96 mmol) in tetrahydrofuran
(8 ml) and water (2 ml) was stirred for 3 h at room temperature.
The solvent was concentrated in vacuo and the resulting oil was
redissolved in water (20 ml). The solution was acidified to pH 2
and a solid precipitated. The solid was filtered through a sintered
glass funnel, washed with a small amount of ether and dried in
vacuo to get the free amino acid 5 (220 mg, 0.40 mmol, 84% yield).
mp: >215 ◦C; Rf: 0.27 (SiO2, dichloromethane/methanol = 9/1);
1H-NMR (400 MHz, [d6]-DMSO) d = 3.48-3.68 (m, 2H, CH2),
In a 50 ml round-bottomed flask Pd/C (20%) and the di-Boc-
mono-Cbz-template 3 (460 mg, 0.79 mmol) were suspended in
MeOH (25 ml). The suspension was vigorously stirred for 1 h
at room temperature under hydrogen atmosphere. The Pd/C
was filtered off through a bed of celite and thoroughly washed
with methanol. The filtrate and washings were combined and
evaporated in vacuo to yield after drying the colorless template
4 (350 mg, 0.79 mmol, 99% yield). mp: 84 ◦C (decomposition);
Rf: 0.61 (RP18, water/methanol/TFA = 2/8/0.1); 1H-NMR
(400 MHz, [d6]-DMSO) d = 1.04-1.12 (m, 9H, CH2-CH3), 1.38
3
3
4.23-4.24 (m, 1H, CH), 5.03 (d, J = 1.1 Hz, 2H, benzyl-CH2),
(s, 18H, Boc-CH3), 2.61-2.74 (m, 6H, CH2-CH3), 3.69 (t, J =
5.14 (s, 2H, benzyl-CH2), 6.78 (m, 1H, pyrrole-CH), 6.97 (s, 1H,
pyrrole-CH), 7.30-7.39 (m, 10H, aryl-CH), 7.58 (d, 3J = 8.1 Hz,
1H, NH), 8.49 (t, 3J = 5.5 Hz, 1H, NH), 8.77 (br.s, 1H, gua-NH),
9.36 (br.s, 1H, gua-NH), 11.85 (br.s, 2H, gua-NH and pyrrole-
NH); 13C-NMR (100 MHz, [d6]-DMSO) d = 40.0 (CH2), 53.9
(CH), 65.5, 66.2 (benzyl-CH2), 112.1, 114.7 (pyrrole-CH), 127.0
(pyrrole-Cq), 127.6, 127.7, 127.8, 127.9, 128.3, 128.4 (aryl-CH),
5.8 Hz, 2H, CH2), 4.17 (d,3J = 4.7 Hz, 4H, CH2), 6.59 (br.s, 2H,
NH2); 13C-NMR (100 MHz, [d6]-DMSO) d = 16.6, 16.8 (CH2-
CH3), 22.5, 22.7 (CH2-CH3), 28.6 (Boc-CH3), 38.5, 39.4 (CH2-
NH), 78.0 (Boc-Cq), 132.3 (Cq-CH2-NH), 142.1, 142.2 (Cq-CH2-
-1
˜
CH3), 155.6 (CO); FT-IR n (KBr-pellet) [cm ] = 3460 [m], 3346
[m], 2973 [s], 2931 [s], 2873 [m], 1706 [s], 1500 [s], 1391 [m], 1365
[s], 1248 [s], 1168 [s], 1046 [m], 1019 [m], 867 [m], 777 [m]; HR-MS
(pos. ESI) m/z = 472.315 0.005 (calculated for C25H43N3O4 +
Na+: 472.315).
130.0 (pyrrole-Cq), 136.6, 136.9 (aryl-Cq), 156.0, 158.9, 160.0,
-1
˜
171.9 (CO and CN); FT-IR n (KBr-pellet) [cm ] = 3347 [m], 2926
[w], 1688 [s], 1650 [s], 1560 [s], 1475 [w], 1290 [s], 1249 [s], 1149
[m], 1045 [w], 750 [m]; HR-MS (neg. ESI) m/z = 549.173 0.005
tert-Butyl ((S)-benzyl 3-(5-(N-(benzyloxycarbonyl)carbamimidoyl-
carbamoyl)-1H-pyrrole-2-carboxamido)-1-oxopropan-2-ylcarba-
mate)-2,4,6-triethyl-1,3-phenylene)bis(methylene)dicarbamate 6
-
(calculated for C26H25N6O8 : 549.174).
Benzyl, di-tert-butyl (2,4,6-triethylbenzene-1,3,5-triyl)tris-
(methylene)tricarbamate 3
In a 50 ml round-bottomed flask pyrrole amino acid 5 (170 mg,
0.31 mmol), PyBOP (161 mg, 0.31 mmol) and DMAP (37.7 mg,
0.31 mmol) were dissolved in DMF (2 ml) to give a yellow solution.
Template 4 (93.0 mg, 0.21 mmol) and NMM (1 ml) in DCM (8 ml)
were added to the reaction mixture. The reaction mixture was
stirred for 24 h at room temperature. The organic layer was con-
centrated using a rotary evaporator. The crude product was first
purified via MPLC (SiO2: cyclohexane/ethyl acetate gradient) and
then via an additional RP18 MPLC (water/methanol gradient)
to get the colorless product 6 (120 mg, 0.12 mmol, 60% yield).
mp: 169 ◦C (decomposition); Rf: 0.30 (RP18, water/methanol =
In a 250 ml three necked flask the triamine 2 (1.42 g, 3.96 mmol)
and sodium hydroxide (0.68 g, 16.9 mmol) were dissolved in a
mixture of dioxane (40 ml) and water (40 ml) to give a yellow
solution. Benzyl chloroformate (1.15 g, 6.74 mmol) and Boc-
anhydride (2.70 g, 12.4 mmol) were added over a period of 4 h to
the cooled (0 ◦C) reaction mixture. The reaction was stirred with
a magnetic stir bar for an additional 2 h at room temperature. The
organic solvent was evaporated and the resulting aqueous solution
was extracted with dichloromethane (3 ¥ 30 ml). The combined
organic layers were washed with brine and dried with MgSO4. The
organic layer was concentrated in vacuo. The crude product was
purified on a triethylamine deactivated silica gel MPLC column
and was eluted with cyclohexane/ethyl acetate gradient to get the
colorless di-Boc-mono-Cbz-template 3 (480 mg, 0.82 mmol, 21%
yield). mp: 67-71 ◦C; Rf: 0.26 (SiO2, cyclohexane/ethyl acetate =
1
2/8); H-NMR (400 MHz, [d6]-DMSO) d = 1.01-1.07 (m, 9H,
CH2-CH3), 1.38 (s, 18H, Boc-CH3), 2.58-2.71 (m, 6H, CH2-CH3),
3.50-3.51 (m, 2H, CH2), 4.17-4.31 (m, 7H, CH and CH2), 5.00
(s, 2H, benzyl-CH2), 5.14 (s, 2H, benzyl-CH2), 6.51 (s, 2H, NH),
6.77 (s, 1H, pyrrole-CH), 7.31-7.40 (m, 11H, aryl-CH and pyrrole-
CH), 7.77 (br.s, 1H, NH), 8.37 (br.s, 1H, NH), 8.80 (br.s, 1H,
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gua-NH), 9.35 (br.s, 1H, gua-NH), 11.15 (br.s, 1H, gua-NH),
12.26 (br.s, 1H, pyrrole-NH); 13C-NMR (100 MHz, [d6]-DMSO)
d = 16.0, 16.2 (CH2-CH3), 22.3, 22.5 (CH2-CH3), 28.2 (Boc-CH3),
37.4, 38.1 (CH2-NH), 54.7 (CH), 65.5 (benzyl-CH2), 77.7 (Boc-
Cq), 112.2, 115.2 (pyrrole-CH), 127.6, 127.7, 128.3, 128.4 (aryl-
CH), 131.3, 132.3 (Cq-CH2-NH), 136.8 (aryl-Cq), 142.8, 143.3
6.78 (m, 1H, pyrrole-CH), 6.98 (s, 1H, pyrrole-CH), 7.30-7.39
(m, 12H, imidazole-CH and aryl-CH), 7.78 (br.s, 1H, NH), 7.84
(br.s, 2H, NH), 8.17-8.20 (m, 2H, Ac-NH), 8.41 (br.s, 1H, NH),
8.80 (s, 1H, gua-NH), 8.95 (s, 2H, imidazole-CH), 9.39 (br.s, 1H,
gua-NH), 11.83 (br.s, 1H, gua-NH), 14.15 (br. s, 2H, imidazole-
NH+); HR-MS (pos. ESI) m/z = 1140.537 0.005 (calculated for
C57H70N15O11+: 1140.537).
˜
(Cq-CH2-CH3), 155.2, 155.6, 159.8, 169.5 (CO and CN); FT-IR n
(KBr-pellet) [cm-1] = 3392 [m], 3301 [m], 2974 [m], 2931 [w], 1685
[s], 1644 [s], 1527 [s], 1267 [s], 1216 [m], 1166 [m], 1046 [w], 757
[w]; HR-MS (pos. ESI) m/z = 1004.485 0.005 (calculated for
C51H67N9O11 + Na+: 1004.485).
N2-((S)-2-Amino-3-(3,5-bis(((S)-2-acetamido-3-(1H-imidazol-4-
yl)propanamido)methyl)-2,4,6-triethylbenzylamino)-3-oxopropyl)-
N5-carbamimidoyl-1H-pyrrole-2,5-dicarboxamide chloride salt 1
(S)-(5-((3-(5-(N-(Benzyloxycarbonyl)carbamimidoylcarbamoyl)-
1H-pyrrole-2-carboxamido)-2-(benzyloxycarbonylamino)pro-
panamido)methyl)-2,4,6-triethyl-1,3-phenylene)dimethanaminium
chloride 7
Compound 8 (145 mg, 0.13 mmol) was dissolved in MeOH (20 ml)
and Pd/C (20%) was added. The suspension was stirred under
hydrogen atmosphere at 40 ◦C for 3 h. The reaction mixture
was separated from the Pd/C through a membrane filter. The
organic solvent was evaporated in vacuo and the resulting solid
was lyophilized twice with hydrochloric acid 0.1 N to yield
the colorless product 1 (120 mg, 0.12 mmol, 93% yield). mp:
220 ◦C (decomposition); Rf: 0.33 (RP18, water/methanol/TFA
Compound 6 (90.0 mg, 0.09 mmol) was dissolved in DCM (2 ml)
and trifluoroacetic acid (2 ml) was added. The reaction mixture
was stirred at room temperature for 3 h. Then the solvents were
removed in vacuo and the resulting oil was dissolved in methanol
and hydrochloric acid (5%) and lyophilized to get the deprotected
template 7 (75.0 mg, 0.09 mmol, 96% yield) as a colorless solid. mp:
>250 ◦C; Rf: 0.37 (RP18, water/methanol/TFA = 2/8/0.1); 1H-
NMR (400 MHz, [d6]-DMSO) d = 1.03-1.10 (m, 9H, CH2-CH3),
2.71-2.79 (m, 6H, CH2-CH3), 3.46-3.65 (m, 2H, CH2), 4.01-4.02
(d, 3J = 4.2 Hz, 4H, CH2), 4.28-4.38 (m, 3H, CH and CH2), 4.95-
5.03 (m, 2H, benzyl-CH2), 5.25 (s, 2H, benzyl-CH2), 6.87 (s, 1H,
pyrrole-CH), 7.32-7.49 (m, 12H, aryl-CH, NH and pyrrole-CH),
7.89 (s, 1H, NH), 8.35 (s, 6H, NH3+), 8.60 (br.s, 1H, gua-NH),
9.32 (br.s, 1H, gua-NH), 9.84 (br.s, 1H, gua-NH), 12.29 (br.s, 1H,
pyrrole-NH); 13C-NMR (100 MHz, [d6]-DMSO) d = 15.8, 15.9
(CH2-CH3), 22.9, 23.2 (CH2-CH3), 36.0, 37.3 (CH2-NH), 41.0
(CH2), 54.5 (CH), 65.5, 67.9 (benzyl-CH2), 113.0, 116.2 (pyrrole-
CH), 127.6 (pyrrole-Cq), 127.8, 128.2, 128.3, 128.5, 128.6, 128.7
(aryl-CH), 129.0 (pyrrole-Cq), 132.4 (Cq-CH2-NH), 135.1, 136.3
1
3
= 2/8/0.1); H-NMR (400 MHz, [d6]-DMSO) d = 1.02 (t, J =
7.04 Hz, 9H, CH2-CH3), 1.81 (s, 6H, Ac-CH3), 2.59 (br.s., 6H,
CH2-CH3), 2.90-3.12 (m, 4H, CH2), 3.61-3.73 (m, 2H, CH2), 4.05
(br.s, 1H, CH), 4.20-4.38 (m, 6H, CH2), 4.61-4.66 (m, 2H, CH),
6.95 (br.s, 1H, pyrrole-CH), 7.34 (s, 2H, imidazole-CH), 7.60 (br.s,
1H, pyrrole-CH), 7.89 (d,3J = 3.00 Hz 2H, NH), 8.30-8.35 (m, 2H,
NH), 8.53-8.73 (m, 8H, gua-NH, NH and NH3+), 8.93 (br.s, 1H,
NH), 9.00 (s, 2H, imidazole-CH), 12.19 (br.s, 1H, pyrrole-NH),
12.34 (s, 1H, gua-NH), 14.39, 14.58 (br. s, 4H, imidazole-NH
and NH+); 13C-NMR (62.5 MHz, [d6]-DMSO) d = 15.9 (CH2-
CH3), 21.4 (CH2-CH3), 22.3 (CH3), 27.0 (CH2), 37.0 (CH2), 45.0
(CH2), 51.4 (CH), 107.5,115.7, 116.3 (CH), 125.7 (pyrrole-Cq),
127.7 (CH), 129.6 (pyrrole-Cq), 131.6 (Cq), 132.2 (Cq-CH2-NH),
133.4 (CH), 136.8 (Cq), 143.5 (Cq-CH2-CH3), 155.6, 159.6, 166.3,
-1
˜
168.9, 169.4 (CO and CN).FT-IR n (KBr-pellet) [cm ] = 3380 [s],
(aryl-Cq), 144.3, 145.2 (Cq-CH2-CH3), 155.7, 159.5, 169.6 (CO
3257 [s], 2974 [m], 1701 [s], 1651 [s], 1550 [s], 1284 [s], 1200 [w],
1079 [w], 763 [w], 626 [w]; HR-MS (pos. ESI) m/z = 894.446
0.005 (calculated for C41H57N15O7 + Na+: 894.445).
-1
˜
and CN); FT-IR n (KBr-pellet) [cm ] = 3365 [s], 2970 [s], 1701 [s],
1655 [s], 1556 [s], 1285 [s], 1189 [m], 754 [m], 698 [w]; HR-MS (pos.
ESI) m/z = 782.399 0.005 (calculated for C41H52N9O7: 782.398).
Benzyl (S)-3-(5-(N-(benzyloxycarbonyl)carbamimidoyl-
carbamoyl)-1H-pyrrole-2-carboxamido)-1-(3,5-bis(((S)-2-
acetamido-3-(1H-imidazol-4-yl)propanamido)methyl)-2,4,6-
triethylbenzylamino)-1-oxopropan-2-ylcarbamate 8
Kinetic measurements
Kinetic data were obtained using a HPLC assay. The general
procedure was to prepare stock solutions of substrate 16, and
compound 1 and benzamide as internal standard in DMSO. The
actual samples were prepared from these stock solutions by mixing
aliquots with the aqueous buffer followed by adjustment of the
pH. Additional amounts of pure DMSO and water were added
to obtain the same solvent compositions for every measurement
(10% DMSO, 1 mM substrate, 20 mM buffer). The time course
measurements were started after addition of substrate 16. The
HPLC runs were performed on an Dionex HPLC system with
P680 pump, ASI-100 auto sampler, and UVD340U diode array
detector. An RP8 phase (Supelcosil 25 cm, 4.6 mm, 5 mm material)
was used to obtain good separation and short retention times.
The elution solvent was 15% MeOH in 10 mM phosphate buffer
solution.
Ac-His-OH (211 mg, 1.07 mmol) was dissolved in DMF (10 ml),
DCM(5 ml) andNMM(1.00ml). To this solution PyBOP(557mg,
1.07 mmol) and DMAP (131 mg, 1.07 mmol) were added and
after a few minutes template 7 (305 mg, 0.36 mmol). The reaction
mixture was stirred at room temperature for 24 h. The organic layer
was concentrated in vacuo. The crude product was purified via
MPLC (RP18; water/methanol gradient) to ◦get the pure product
8 (155 mg, 0.14 mmol, 38% yield). mp: 205 C (decomposition);
1
Rf: 0.26 (RP18, water/methanol = 2/8); H-NMR (400 MHz,
[d6]-DMSO) d = 1.01-1.02 (m, 9H, CH2-CH3), 1.79 (s, 6H, Ac-
CH3), 2.58 (br, 6H, CH2-CH3), 2.85-3.07 (m, 4H, CH2), 3.51
(br.s, 2H, CH2), 4.19-4.35 (m, 6H, CH2), 4.59-4.64 (m, 2H, CH),
4.97-4.99 (m, 2H, benzyl-CH2), 5.15 (s, 2H, benzyl-CH2), 6.76-
4902 | Org. Biomol. Chem., 2009, 7, 4895–4903
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6 J. Razkin, J. Lindgren, H. Nilsson and L. Baltzer, ChemBioChem, 2008,
9, 1975.
Acknowledgements
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We thank the DFG (Deutsche Forschungsgemeinschaft) and the
Fonds of the Chemical Industry for ongoing financial support of
our work and Dr. H.-G. Korth (university of Duisburg-Essen) for
helpful comments.
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The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 4895–4903 | 4903
©