Probing the effect of the amidinium group and the phenyl ring on the thermodynamics of binding of benzamidinium chloride to trypsin

Article abstract of DOI:10.1039/b410061a

The effect of the amidinium group and the phenyl ring on the thermodynamics of binding of benzamidinium chloride to the serine proteinase trypsin has been studied using isothermal titration calorimetry. Binding studies with benzylammonium chloride, α-meth

Full text of DOI:10.1039/b410061a

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A R T I C L E
Probing the effect of the amidinium group and the phenyl ring on the
thermodynamics of binding of benzamidinium chloride to trypsin
Reinskje Talhout and Jan B. F. N. Engberts*
Physical Organic Chemistry Unit, Stratingh Institute, University of Groningen, Nijenborgh 4,
9747 AG Groningen, The Netherlands. E-mail: j.b.f.n.engberts@chem.rug.nl;
Fax: +31 (0)50 3634296; Tel: +31 (0)50 3634242
Received 5th July 2004, Accepted 7th September 2004
First published as an Advance Article on the web 30th September 2004
The effect of the amidinium group and the phenyl ring on the thermodynamics of binding of benzamidinium
chloride to the serine proteinase trypsin has been studied using isothermal titration calorimetry. Binding studies
with benzylammonium chloride, a-methylbenzylammonium chloride and benzamide, compounds structurally
related to benzamidinium chloride, showed that hydrogen bonding between the amidinium group and the enzyme
is primarily enthalpy-driven. Binding of cyclohexylcarboxamidinium chloride and acetamidinium chloride showed
that the hydrophobic binding of the phenyl ring in the S1 pocket is primarily entropy-driven and that a rigid, flat
hydrophobic binding site for the inhibitor is favourable. The compounds that have been studied over a range of
temperatures exhibit a negative change in heat capacity upon binding and enthalpy–entropy compensation, both
characteristic of hydrophobic interactions.
Introduction
The guanidinium–carboxylate binding mode, with its strong
binding interactions, is ubiquitous in enzyme–substrate binding
as well as in the stabilisation of protein tertiary structures via
internal salt bridges.1 The serine proteinase trypsin specifically
cleaves the peptide bonds on the carboxyterminal side of the
positively charged residues arginine and lysine, which bind in the
specificity pocket to the negatively charged Asp189 carboxylate.
Non-transition state inhibitors block binding of the substrate
by binding in the specificity pocket. Many natural proteinase
inhibitors are small peptides with an arginine or lysine residue
binding in the specificity pocket and synthetic proteinase inhibi-
tors are also often based on these interactions.2 Regarding the
specificity requirements of the enzyme, it has been reported in
1965 that amidines are good model systems for the arginine
side chain of trypsin substrates.3 Benzamidinium chloride and
p-aminobenzamidinium chloride were the most potent small
molecular competitive inhibitors of trypsin reported until then.
Apparently, the phenyl group is a good model for the hydro-
phobic part of the arginine side chain. The lengths of these
groups are approximately the same, but the rigid phenyl group
has only one single conformation.
In this study, we investigated the influence of the amidinium
group and the phenyl ring on the thermodynamics of binding of
benzamidinium chloride to trypsin. To this purpose, the bind-
ing of the structurally related compounds benzylammonium
chloride, a-methylbenzylammonium chloride, benzamide, cyclo-
hexylcarboxamidinium chloride, and acetamidinium chloride,
was examined by means of isothermal titration calorimetry
(ITC).
Fig. 1a shows a cartoon representation of the crystal structure
of the benzamidinium–trypsin complex.4 The benzamidinium
ion is bound in the specificity pocket S1, making five hydrogen
bonds with Asp189, Gly219, Ser190 and an internal water
molecule.
Both partially positively-charged amidinium nitrogens bind
to the carboxylate oxygens of Asp 189 in an almost symmetri-
cal manner, N1 to Asp189 OD1 (2.90 Å) and N2 to Asp189
OD2 (3.12 Å). In addition, N1 is hydrogen bonded to Gly219
O (2.83 Å), while N2 is hydrogen bonded to both Ser 190 OG
(3.06 Å) and the internal water molecule 416 (3.16 Å). The phe-
nyl ring is mainly surrounded by non-polar and neutral polar
a-amino acid residues and is in van der Waals contact with the
residues 214–216 and 190–191.5
Fig. 1 Cartoon representations of the crystal structures of the benzam-
idinium–trypsin complex4 (a), and the benzylammonium–trypsin com-
plex7 (b) (3ptb and 2bza from the Brookhaven Protein Data Bank,29
respectively) generated using the program RasMol.30 Both inhibitors
are bound in the S1 binding pocket, close to the catalytic triad (His57,
Asp102, Ser195).
Just as amidinium ions are good structural analogues of
the arginine side chain, alkyl ammonium ions are good model
T h i s j o u r n a l i s
©
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 1 – 3 0 7 4
3 0 7 1

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Table 1 Thermodynamic parameters for binding of benzylammonium chloride, cyclohexylcarboxamidinium chloride, and benzamidinium chloride
to trypsin in Tris buffer at pH 8.0 at different temperatures
Compound (chloride salt)
Benzylammonium
T/°C
K/104 M−1
DG/kJ mol−1
DH/kJ mol−1
TDS/kJ mol−1
20.1
25.1
30.0
37.1
20.1
25.2
30.1
37.0
20.1
25.1
30.0
37.1
0.71
0.55
0.50
0.42
0.42
0.34
0.32
0.28
6.2
−21.6
−21.4
−21.5
−21.5
−20.3
−20.2
−20.4
−20.4
−26.9
−26.6
−26.7
−26.7
−8.5
−9.9
13.1
11.5
10.5
8.6
3.3
0.8
−1.1
−4.4
9.9
7.7
5.4
−11.0
−12.9
−17.0
−19.4
−21.5
−24.8
−17.0
−18.9
−21.4
−23.7
Cyclohexylamidinium
Benzamidiniuma
4.5
4.1
3.1
3.0
a Data taken from ref. 10.
systems for lysine.6 The benzylammonium ion was found to
be the most efficient inhibitor of the compounds investigated,
followed by the n-butylammonium ion with a three-fold lower
affinity. Since the hydrophobic parts of these ions have similar
lengths, the higher affinity of the phenyl ring is likely due to
other specific properties of the phenyl ring, such as its aromatic
and conformationally restricted character.
formational entropy.11 Therefore, the less favourable enthalpy of
binding is most likely due to less strong interactions with the en-
zyme due to the less extensive hydrogen bonding in comparison
to benzamidinium. The more favourable entropy of binding is
most probably due to enthalpy–entropy compensation, which is
the phenomenon that small structural variations in an inhibitor
often result in changes in the enthalpy and entropy of binding
that are considerably larger than those in the Gibbs energy. Such
enthalpy–entropy compensation is common when studying
weak interactions and is important for understanding the na-
ture of competing interactions.12–14 Compensation is the natural
outcome of the fact that bound species will necessarily have less
freedom of motion in a tighter complex.15,16 We therefore pro-
pose that benzylammonium is less extensively conformationally
restrained in the complex due to the less tight binding.
Concerning the binding to trypsin, it is obvious that the geo-
metry of an amidinium group is more favourable than that of
an ammonium group, which is not able to fully utilise all the
hydrogen-bonding acceptor sites provided by the enzyme. In
comparison to benzamidinium, benzylammonium is expected
to have less effective hydrogen-bonding interactions because of
the absence of one of the amino groups. This is indeed seen in
the crystal structure of the benzylammonium–trypsin complex:7
the cartoon representation in Fig. 1(b) shows a binding mode
different from that of benzamidinium–trypsin. A slight expan-
sion of the binding pocket, involving residues 190–192 and
215–216, has been observed. The ammonium group exclusively
occupies only one of the two possible locations, namely that cor-
responding to the N1 position of benzamidinium; around the
position corresponding to the N2 position of benzamidinium,
no electron density was observed for benzylammonium.8 The
ammonium group forms hydrogen bonds with Asp189 OD1
(2.72 Å) and Ser190 O (2.67 Å), but not with Ser190 OG or
Gly219 O, as is the case for benzamidinium–trypsin. Further-
more, it forms a hydrogen bond (2.99 Å) to a water molecule in-
serted between and forming hydrogen bonds with Asp189 OD1
(3.15 Å) and Gly219 O (3.12 Å). This hydrogen-bonding pattern
may force the nitrogen of the ammonium group of benzylammo-
nium into one specific position.
Over a relatively narrow temperature range, the temperature
dependence of DH is given by:
dDH/dT = DCp
where DCp is the heat capacity change upon binding. For benzyl-
ammonium chloride, the value of DCp, determined from the
slope of the linear fit to the data points, is equal to −254 J mol−1
K−1 (Table 2). A negative value of DCp often correlates with the
burial of non-polar surface area from water.13,17,18 By contrast,
the removal of polar surfaces from the aqueous phase tends to
increase DCp. We contend that the DCp of benzylammonium
chloride is less negative than that of benzamidinium chloride
(−400 J mol−1 K−1, Table 2) because of the fact that the ammo-
nium group has a higher charge density than the amidinium
group.1 Since the removal of polar surfaces from the aqueous
phase tends to increase DCp, the less negative DCp could be
caused by the more polar ammonium group in the benzylammo-
nium ion.
A negative DCp results in a shift in the net thermodynamic
driving force for association from being entropic to enthalpic
with increasing temperature. Temperatures TH and TS can be
defined, at which the enthalpic and the entropic contributions
to binding are zero. Provided that DCp is independent of tem-
perature, together with TH and TS it gives a complete thermo-
dynamic description of the binding process. TH and TS have been
calculated from a linear fit to the temperature-dependencies of
DH and TDS and are listed in Table 2.
Results and discussion
Effect of the amidinium group
In order to probe the contribution of the amidinium group to
the binding of benzamidinium-based inhibitors to trypsin, the
binding of benzylammonium9 chloride to trypsin was studied.
The thermodynamic parameters obtained in Tris buffer at pH
8.0 are listed in Table 1. The thermodynamics of binding of
benzamidinium chloride to trypsin have been published before,10
and are, for the sake of comparison, listed in Table 1 as well.
At all temperatures studied, the binding constant of benzyl-
ammonium chloride to trypsin is more than six times lower than
that of benzamidinium chloride (Table 1) and DG is between 4.7
and 5.0 kJ mol−1 less favourable. This less favourable DG results
from a more than two times less favourable DH that is partly
compensated by a more favourable TDS. It has been suggested
that the enthalpy of binding primarily reflects the strength of
the interactions of the inhibitor with the enzyme, whereas the
entropy change mainly reflects two contributions: changes in
hydration entropy of inhibitor and enzyme and changes in con-
DG is practically temperature-independent as a result of
the compensating temperature-dependencies of DH and TDS
(Table 1). This thermodynamic behaviour is characteristic for
processes accompanied by a large, negative DCp and thus, again,
a consequence of the vanishing of the hydrophobic hydration
of the inhibitor. A negative DCp and enthalpy–entropy com-
pensation with temperature are the main fingerprints indicating
that the hydrophobic effect is involved in biological or chemical
processes.19–21
3 0 7 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 1 – 3 0 7 4

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Table 2 DCp, TS and TH for binding of benzylammonium chloride,
cyclohexylcarboxamidinium chloride, and benzamidinium chloride to
trypsin in Tris buffer at pH 8.0
cannot exclude that some adjustment of the binding pocket may
be necessary to accommodate the cyclohexyl ring.
For cyclohexylcarboxamidinium chloride, DCp is equal
to −458 J mol−1 K−1, TH is −17 °C and TS is 27.4 °C (Table 2).
This large and negative DCp results in enthalpy–entropy com-
pensation with temperature. The heat capacity change upon
binding is slightly higher than that of benzamidinium chloride,
probably due to the fact that the cyclohexyl ring is more hydro-
phobic than the phenyl ring. This is indicated by its higher
n-octanol–water partition coefficient; DlogP, the relative n-oc-
tanol–water partition coefficient of a substituent with respect to
hydrogen, amounts to 2.82 for cyclohexyl and 1.96 for phenyl.22
Compound (chloride salt)
DCp/J mol−1 K−1
TS/°C
TH/°C
Benzylammonium
Cyclohexylamidinium
Benzamidiniuma
−254
−458
−400
71
−17
44
14
27.4
−23
a Data taken from ref. 10.
To even further disturb the hydrogen bonding capacities of
the amidinium group, the binding affinities of a-methylbenzyl-
ammonium and benzamide to trypsin have been tested. For both
compounds, only peaks of dilution were observed at 25 °C when
titrated in trypsin solution, which indicates that binding of these
compounds to trypsin is negligible.
Conclusions
The effect of the amidinium group and the phenyl ring on the
thermodynamics of binding of benzamidinium chloride to tryp-
sin has been investigated by means of isothermal titration calo-
rimetry measurements. The influence of the amidinium group
on the binding process has been probed by comparison with
benzylammonium chloride. The lowered binding affinity of ben-
zylammonium chloride is due to a less favourable enthalpy of
binding that is partly compensated by a more favourable entropy
of binding. This is in accord with the less extensive hydrogen-
bond interactions observed in the benzylammonium–trypsin
complex as compared to the benzamidinium–trypsin complex.
If the amidinium group is even further distorted, as in a-methyl-
benzylamine and benzamide, the binding affinity is negligible.
The influence of the phenyl ring on the binding process
has been probed by considering cyclohexylcarboxamidinium
chloride and acetamidinium chloride. The lowered binding af-
finity of cyclohexylcarboxamidinium chloride is due to a less
favourable entropy of binding, whereas the enthalpy of binding
is rather similar. This is probably due to the restricted confor-
mational freedom of the cyclohexyl ring in the binding pocket.
Acetamidinium chloride, with its reduced hydrophobic area, is
an inefficient inhibitor.
These experiments point out that both the hydrogen-bond
donating amidinium group and the hydrophobic phenyl ring
of the benzamidinium molecule contribute to the binding po-
tency of the benzamidinium molecule to trypsin. Interactions
of the amidinium group are primarily enthalpically favourable,
whereas the interactions of the phenyl ring appear to be pri-
marily entropically favourable. For those compounds measured
over a range of temperatures, the observed thermodynamics of
binding were characteristic of the hydrophobic effect: a large,
negative DCp and, as a consequence, strong enthalpy–entropy
compensation with temperature.
Influence of the phenyl ring
In order to probe the contribution of the phenyl ring to the bind-
ing of benzamidinium-based inhibitors to trypsin, titrations of
acetamidinium chloride and cyclohexylcarboxamidinium chlo-
ride into trypsin were performed. In acetamidinium chloride, the
relatively large and hydrophobic phenyl group has been replaced
by the smaller and less hydrophobic methyl group, which is ex-
pected to have less favourable interactions with the hydrophobic
S1 pocket. The upper limit of the binding constant of acetamidi-
nium chloride to trypsin at 25 °C is 102; more reliable binding
constants are not retrievable for this poorly binding inhibitor.10
This value is significantly lower than the binding constant of
benzamidinium to trypsin (K = 4.5 × 104 M−1 at 25 °C, Table 1),
indicating that the more hydrophobic phenyl group increases the
binding constant at least 450 times (DG is more than 15 kJ mol−1
more favourable) relative to a methyl group.
In cyclohexylcarboxamidinium chloride, the phenyl group
is replaced by the more hydrophobic, but also larger (six addi-
tional C–H bonds) and more flexible cyclohexyl group.22–24 The
polarisabilities of cyclohexane and benzene are, respectively,
10.99 and 10.39 10−24 cm3 (20 °C), which suggests that the disper-
sion interactions of both compounds will be comparable.
As described in the Introduction, the phenyl ring of the benza-
midinium ion sits really tightly in the binding pocket of trypsin,
in closer than van der Waals contact with some hydrophobic
residues of the enzyme. Therefore, for binding of the cyclohexyl-
carboxamidinium ion to trypsin to occur, it may be necessary to
disturb the conformational equilibrium of the cyclohexyl ring
and/or the conformation of the binding site in order to fit the
cyclohexyl ring into the binding pocket. The thermodynamic
binding parameters of cyclohexylcarboxamidinium chloride
to trypsin are listed in Table 1. At all temperatures studied, the
binding constant of cyclohexylcarboxamidinium chloride to
trypsin is more than ten times lower than that of benzamidinium
chloride and DG is more than 6 kJ mol−1 less favourable. This is
an entropic effect and the enthalpy of binding is slightly more
favourable at all temperatures studied.
Experimental
General remarks
Trypsin solutions were prepared and titration experiments per-
formed and analysed as described previously.10, 26 Measurements
were repeated at least three times; K was reproducible to within
10% and DH was reproducible to within 5%. Errors in DCp
were within 5%. Bovine pancreatic trypsin was obtained from
Fluka. The inhibitors were of the highest purity available and
purchased from Aldrich (benzylamine hydrochloride, a-methyl-
benzylamine, benzamide), Fluka (acetamidine hydrochloride)
and Sigma (benzamidine hydrochloride). Starting materials for
the synthesis were from Aldrich (cyclohexylcarbonitrile, 2 M
In general, upon binding both the inhibitor and the enzyme
lose conformational freedom, which is an entropic effect. To
reduce the loss in conformational freedom, a strategy in inhibi-
tor design is to conformationally constrain the inhibitor.11,25 If
the free inhibitor itself has little conformational freedom, the
entropy loss upon binding to the enzyme will be small. We
therefore propose that the less favourable entropy of binding of
the cyclohexylcarboxamidinium ion is primarily due to the re-
duced flexibility of the cyclohexyl ring in the binding site of the
enzyme as compared to that in the aqueous phase. This loss in
conformational freedom is not encountered for the already flat
and constrained phenyl ring. Also, the enzyme may lose some
conformational freedom to adjust the binding site for the most
favourable binding of the cyclohexyl group. Furthermore, we
1
solution of trimethylaluminium in toluene). H-NMR spectra
were recorded on Varian Gemini 200 (200 MHz) and VRX 300
(300 MHz) spectrometers. Elemental analyses were performed in
the analytical department of our laboratory by Mr E. Brussee.
Synthesis of cyclohexylcarboxamidinium chloride
Cyclohexylcarboxamidinium chloride was synthesised as
outlined in Scheme 1. An adapted literature procedure27 was
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 1 – 3 0 7 4
3 0 7 3

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8 The low temperature factor (ca. 20 Å) is another indication that only
a single conformation of the benzylammonium ion is present in the
binding pocket. Interestingly, molecular dynamics simulations on the
benzylammonium–trypsin complex showed two different conforma-
tions of benzylammonium in the pocket. One exactly matches the
electron density map of the benzylammonium–trypsin crystals and
the other forms two hydrogen bonds with the carbonyl oxygen of
Ser190 and Asp189 OG2.
9 Since the pKa of benzylammonium is 9.5 at pH 8.0 (C. H.
Arrowsmith, H.-X. Guo and A. J. Kresge, J. Am. Chem. Soc.,
1994, 116, 8890–8894), the pH at which the measurements are
performed, 3.1% of the ammonium group is deprotonated, which
is not considered significant. The percentage was calculated using
the Henderson–Hasselbalch equation (A. Fersht, Structure and
Mechanism in Protein Science, Freeman, New York, 1999):
used. A 2 M solution of Me3Al in toluene (15.3 ml, 30.5 mmol)
was slowly added to a magnetically stirred suspension of 1.8 g
(33 mmol) of NH4Cl in 14 ml of toluene (suspension dried by
azeotropic distillation to 14 ml of 27 ml of toluene under a N2
atmosphere) at 0 °C under a N2 atmosphere. After the addition,
the mixture was warmed to 25 °C and stirred until the gas evolu-
tion had ceased. Cyclohexylcarbonitrile (2.0 g, 18 mmol) was
added and the solution was heated to 80 °C under a nitrogen
atmosphere until TLC indicated the absence of nitrile. The reac-
tion mixture was slowly poured into a slurry of 10 g silica gel in
30 ml of chloroform and stirred for 5 min. Next, 25 ml methanol
was added and the suspension was stirred for 2 h. The silica was
filtered off and washed with methanol. The filtrate and wash
were combined and stripped to a residue of 17 ml, which was
filtered to remove the precipitated NH4Cl. Finally, the solvent
of the filtrate was evaporated.
[B]
[BH+
pH=pKa +log
]
where [B] is the concentration of deprotonated benzylammonium
and [BH+] is the concentration of benzylammonium.
10 R. Talhout and J. B. F. N. Engberts, Eur. J. Biochem., 2001, 268,
1554–1560.
11 S. Leavitt and E. Freire, Curr. Opin. Struct. Biol., 2001, 11, 560–
566.
12 C. T. Calderone and D. H. Williams, J. Am. Chem. Soc., 2001, 123,
6262–6267.
13 A. Cooper, C. M. Johnson, J. H. Lakey and M. Nöllmann, Biophys.
Chem., 2001, 93, 215–230.
14 J. D. Dunitz, Chem. Biol., 1995, 2, 709–712.
15 W. C. Conner, J. Catal., 2003, 78, 238–246.
16 S. W. Benson, Thermochemical Kinetics, Wiley: New York, 1968.
17 Structure-Based Drug Design: Thermodynamics, Modelling and
Strategy, ed. J. E. Ladbury, and P. R. Connelly, Springer: Berlin,
1997.
18 Protein–Ligand Interactions: Hydrodynamics and Calorimetry, eds.
S. E. Harding, and B. Z. Chowdry, Oxford University Press, Oxford,
2001.
19 W. Blokzijl and J. B. F. N. Engberts, Angew. Chem., Int. Ed. Engl.,
1993, 32, 1545–1579.
20 N. T. Southall, K. A. Dill and A. D. J. Haymet, J. Phys. Chem. B,
2002, 106, 521–533.
21 J. H. Ha, R. S. Spolar and M. T. Record, J. Mol. Biol., 1989, 209,
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22 C. Hansch; A. Leo in Exploring QSAR: Fundamentals and Applica-
tions in Chemistry and Biology, ed. S. R. Heller, ACS, Washington,
DC, 1995.
23 G. Graziano, Can. J. Chem., 2000, 78, 1233–1241.
24 W. P. Jencks, Catalysis in Chemistry and Enzymology, McGraw-Hill,
New York, 1969, pp. 393–436.
Scheme 1 Synthesis of cyclohexylcarboxamidinium chloride.
The crude product was purified by stirring overnight in 10 ml
of i-propanol, which was filtered to remove the precipitated
NH4Cl and the solvent of the filtrate was evaporated. To remove
organic contaminations, the product was dissolved in as little i-
propanol as possible and titrated in 300 ml of ether, from which
the precipitate was filtered off; this procedure was performed
twice. The product was washed with ether and n-hexane and sub-
sequently freeze-dried from 1 M hydrochloric acid to yield 1.8 g
(60%) of cyclohexylcarboxamidinium chloride,28 mp 181–182 °C
(dec., lit.28 184–185 °C). 1H-NMR (300 MHz, DMSO-d6, ppm):
8.92 (s, broad, 2H), 8.81 (s, broad, 2H), 2.44 (t, 1H, J = 12.3 Hz),
1.78–1.10 (m, 10H). Elemental analysis: calc: C 51.69% H 9.29%
N 17.22% found: C 51.63% H 9.43% N 17.23%.
25 M. J. Todd, I. Luque, A. Velásquez-Campoy and E. Freire, Biochem-
istry, 2000, 39, 11876–11883.
26 R. Talhout, A. Villa, A. E. Mark and J. B. F. N. Engberts, J. Am.
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M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi and
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3 0 7 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 7 1 – 3 0 7 4