Concentration and basicity of histamine rotamers

Article abstract of DOI:10.1039/b200846g

A method was developed and applied to determine the populations and site- and conformer-specific basicities of histamine rotamers in three distinct states of protonation. The method that has been developed also allows the determination of molecule- and po

Full text of DOI:10.1039/b200846g

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Concentration and basicity of histamine rotamers
Márta Kraszni, József Kökösi and Béla Noszál*
Semmelweis University, Department of Pharmaceutical Chemistry, H-1092 Budapest,
Högyes E. u. 9, Hungary. E-mail: NOSBEL@hogyes.sote.hu; Fax: ϩ361-2170891
2
Received (in Cambridge, UK) 23rd January 2002, Accepted 27th February 2002
First published as an Advance Article on the web 22nd March 2002
A method was developed and applied to determine the populations and site- and conformer-specific basicities of
histamine rotamers in three distinct states of protonation. The method that has been developed also allows the
determination of molecule- and position-specific standard gauche and trans 1H NMR coupling constants, and
the subsequent rotamer analysis from NMR spin systems with a single observed coupling constant, introducing
simplifying allowances of symmetry origin. Synthesis and NMR analysis of a reference histamine derivative
have also been carried out. The trans rotamers of histamine were found to exist in 41, 38 and 50% in the neutral,
monocationic and dicationic forms, respectively, providing quantitative, experimental evidence for the preferential
thermodynamic properties of the trans species over the two enantiomeric, statistically favoured g conformers in any
ionisation form. Quantification of the rotamer- and site-specific basicities resulted in increased amino and decreased
imidazole basicities in the gauche rotamer, indicating the formation of intramolecular hydrogen bonds in the
monocationic form.
occurs at 80% in the N(τ)–H tautomer.8,9 At lower pH (still
present in living systems) the dicationic species predominates.
Introduction
Histamine, the small, versatile biogenic amine is one of the
most thoroughly studied chemical entities, due to its enormous
biological significance. Its biochemical functions are mediated
mainly via histamine receptors,1,2 of which four different types
have been identified so far. The exogenous, specific H1, H2, etc.
agonists and antagonists are ligands of one particular receptor
only, unlike histamine, the endogenous activator of every
histamine receptor. This indicates that histamine binds to the
H1, H2, etc. receptors in different conformations and/or forms
of ionisation. Characterisation of the various conformational,
ionisable forms is therefore an important step in elucidating the
molecular background to the biological versatility.
Solid phase X-ray crystallographic studies proved the presence
of the trans conformation in all three protonation states.10–12
Nevertheless, the trans and gauche conformers coexist in solu-
tion, and their ratios in the monocationic and neutral forms
1
have been found to be nearly equal,13,14 as determined by H
NMR spectroscopy in D2O. Theoretical studies have also been
carried out to predict preferential conformations in the gas
phase and solution.13,15–19 The reported values reflect method-
dependent energy differences, in which ab initio and Monte
Carlo studies18 showed preferential gauche conformers,
whereas AMBER force field methods19 resulted in a higher
concentration of the trans rotamer.
Despite its mere 8 skeleton atoms, histamine occurs in a
variety of structural forms.3 The amino and imidazole moieties
are differently and interactively basic, i.e. protonation at
one site significantly reduces the basicity of the other site. The
molecule therefore takes up several forms of protonation. The
imidazole ring undergoes tautomeric changes, the extent of
which depends on the molecular state of ionisation. Also, the
imidazole C(2) proton is one of the few carbon bonded acidic
protons.4 Histamine is considered an achiral, prochiral com-
pound. Its prochirality becomes evident if any of the methylene
geminal hydrogens is substituted. Such substitution can be
a histamine histidine conversion, or simply, a deuterium–
hydrogen replacement. Equivalence of the geminal hydrogens
results in identical concentrations of stereochemically distinct
species, which allows simplified treatment of some conformer-
specific parameters, as shown in the Results and discussion
section. The sigma bonds in the aliphatic side-chain permit
rotation, giving rise to different conformations of the molecule.
Due to the above properties, histamine exists in solution as a
mixture of several ionic, tautomeric and conformational forms
of short individual lifetime and rapid interconversion. These
species, however, have their own physicochemical properties,
and they act individually and specifically in stereochemically
controlled biochemical processes.
The H1, H2, H3 and H4 receptors and their selectivity towards
exogenous ligands assume four different, active conform-
ations of histamine. Their binding stereochemistry could be
characterised by docking studies from knowledge of the three-
dimensional (3D) structures of the receptor pockets. Although
significant progress has been achieved in elucidating the amino
acid sequence and other molecular properties of histamine
receptors,20–23 the 3D structures have not yet been reported.
Conclusions on the binding forms of histamine can therefore be
drawn from structure-dependent activity investigations of syn-
thetic histamine derivatives, including the exogenous agonists
and antagonists. Such investigations suggest the fitting of the
extended trans–trans conformation of the N(τ)–H histamine
monocation to H1 and H2 receptors.24–28 The capability of
undergoing 1–3 prototropic tautomerism was assumed to be
necessary for H2 activity.26 Much less is known about the
stereochemical requirements on the H3 receptor, but existence
of the gauche–trans conformer has been hypothesised.28,29 No
data has appeared on the stereochemical aspects of the
histamine–H4 complex. The stereochemistry of histamine
species is also important in metal complexation,30 and heparin-
bound storage in mast cells.31
Since the biological function of any of the species cannot be
ruled out, and the reactive species is not necessarily the major
one,4 we determined the concentration of every significant,
coexisting histamine species in aqueous solution. Rotamer
populations were determined for the three protonation states by
The protonation equilibria of histamine have been described
at the macroscopic5,6 and microscopic levels.7 In aqueous solu-
tion at 37 ЊC and physiological pH (7.4) the major species bears
one extra proton at the amino site, while the imidazole ring
1
an improved, molecule-specific version of H NMR coupling
914
J. Chem. Soc., Perkin Trans. 2, 2002, 914–917
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b200846g

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constant analysis. The acid–base properties of every conformer
have also been quantified.
and position-specific trans and gauche constants, calculated
by means of moiety electronegativities introduced by Altona
et al.38
1
The H NMR spectrum of histamine contains the inform-
Experimental
ation of a single observed vicinal coupling value only. Rotamer
populations could thus be obtained by a modified version of
the analysis, in which molecular symmetry has been taken into
account in the process of evaluation.
Histamine dihydrochloride, deuterium chloride, and sodium
deuteroxide were obtained from Sigma Chemical Co. Deuter-
ium oxide was obtained from Merck. The 0.1 M histamine solu-
tions were prepared in 10% D2O and 90% H2O mixtures, 2 M
DCl–HCl and 2 M NaOD–NaOH 10 : 90 solutions were used
to set the pH.
The four aliphatic protons of histamine form an AAЈBBЈ
spin system where A and AЈ, B and BЈ protons are chemically
equivalent. Rotation about the α–β carbon–carbon bond is
rapid on the NMR time-scale. The AAЈBBЈ spin system
appears as two symmetrical triplets, indicating the identical
NMR behaviour of conformers g and gЈ, the mirror image
species. Thus, the conformational equilibrium of histamine can
be characterised by the ratio of two rotamers, where the occur-
rence probability of rotamer g is two times higher than that of
rotamer t.
pH Measurements were carried out with a Radiometer
PHM93 pH meter, equipped with an Orion 9103BN semi micro
combined glass electrode. The electrode was calibrated using
1
aqueous (pH = 4.003 and 9.198, 296 K) buffer solutions. H
NMR studies were carried out using Bruker AM 360 and
Bruker AM 200 spectrometers. Protonation constants of his-
tamine were determined by 1H NMR–pH titrations in solutions
of 16 different pH values, ranging from 3.5 to 13, and at 296 K.
Chemical shifts were measured relative to internal tert-butyl
alcohol (1.236 ppm). For the determination of the vicinal
proton–proton coupling constants of histamine, NMR spectra
were recorded at pH = 3.5, 8.0, and 13, where the three distinct
protonation states of the molecule exist almost exclusively.
Coupling constants were obtained from spectra of 0.02 Hz
digital resolution.
The observed vicinal proton–proton coupling constant of
histamine is a weighted sum of the various gauche and trans
coupling constants of individual rotamers, where weighting
factors are the appropriate mole fractions:
(1)
For the determination of imidazole substituent constants,
4-isopropylimidazole was synthesised. As a first step 1-bromo-
3-methylbutan-2-one was prepared from 3-methylbutan-2-one
(Fluka) by the method of McMorris et al.32 This substance
was then condensed with formamidine acetate to form
4-isopropylimidazole in a similar manner to the literature
method.33,34 After isolation and purification of the product by
TLC, its structure was proved by NMR. H NMR coupling
constants of 4-isopropylimidazole were recorded in D2O at pD
< 2 and pD > 10.
αt ϩ αg = 1
(2)
3
In eqns. (1)–(2), JHH is the observed coupling value of his-
tamine, αt and αg are rotamer populations, JT is the standard
coupling constant corresponding to a 180Њ dihedral angle, JGt,
JGg , JGg and JGg are standard coupling constants corre-
sponding BtЈAo 60Њ andAB300Њ dihedral angles in rotamers t and g,
AЈBЈ
1
respectively, as shown in Fig. 1.
By combining eqns. (1)–(2) we obtain:
Results and discussion
(3)
Rotamer populations
Fig. 1 shows the numbering of the histamine molecule and its
Eqn. (3) shows that the determination of rotamer popu-
lations uses the standard coupling constants. Such parameters
are not ’a priori’ known. In order to determine their molecule-
and species-specific values we calculated the JT, JGt, JGg
,
AЈBЈ
JGg and JGg quantities from the reparametrised Karplus
equation of AAlBtona et al.38 (Table 1). Using this equation and
empirical group electronegativities (substituent constants) we
could take not only the type of group, but also the effect of its
protonation into consideration. Note that this method provides
four gauche and one trans standard constant for every proton-
ation state. Calculation of the different JTt and JTg parameters,
which exist theoretically, resulted in identical values. Substitu-
ent constants have been reported by Altona et al.38 for the
amino and ammonium groups. No analogous literature data
was available for the imidazole and imidazolium sites. We there-
fore measured the coupling constants of 4-isopropylimidazole.
This compound was suggested for such studies by Altona, since
the coupling constants of isopropyl derivatives are independent
of the torsion angles. The values obtained for 4-isopropyl-
imidazole are J = 6.90 Hz for the basic form, and J = 6.93 Hz for
the protonated form. Calculations for the imidazole ring with a
side-chain in the C(5) position resulted in substituent constants
0.50 and 0.47 for the neutral and cationic forms of imidazole,
respectively. The slight difference can be interpreted by the
enhanced electron-withdrawing effect of the imidazolium
moiety, and also by the fact that the positive charge spreads
over the ring.
BЈA
Fig. 1 Histamine: numbering and designation of its atoms and the
staggered forms of its rotamers.
staggered conformers, where the rotational axis is the α–β
carbon–carbon bond. Rotamer analyses have been carried
out on the basis of the Karplus relationship and vicinal
coupling constants.35–37 The vast majority of such analyses
used the observed 1H NMR coupling data on spin systems
with 3 coupling protons, and two observed non-identical
vicinal coupling values. Such data sets allowed the calculation
of the rotamer populations using standard, trans and gauche
coupling constants irrespective of the type of molecule.35–37
As an improvement on rotamer analysis we used molecule-
The standard coupling constants were obtained by the
Altona equation (rms error: 0.36 Hz).38 This allowed the calcu-
J. Chem. Soc., Perkin Trans. 2, 2002, 914–917
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Table 1 Observed and standard vicinal 1H–1H NMR couplings and rotamer populations of histamine
Standard trans and gauche coupling constantsa/Hz
Rotamer
populations
Protonation state
Observed couplings 3JHH/Hz
JT
JGt
JGgAЈBЈ
JGgBЈA
JGgAB
αt
αg
NH2–Im
6.84
6.97
7.35
13.19
13.57
13.62
4.19
4.05
4.04
2.76
3.22
3.21
2.45
2.90
2.93
3.88
3.73
3.76
0.41
0.38
0.50
0.59
0.62
0.50
NH3ϩ–Im
NH3ϩ–Imϩ
a Calculated by Altona’s equation (rms = 0.36 Hz).38
Table 2 Parameters of the non-linear parameter fitting
lation of the rotamer mole fractions according to eqns. (2)–(3).
Error-proliferation calculations resulted in 0.07 and 0.10
uncertainties in the αt and αg values, respectively. Table 1
shows that concentration of the g conformer exceeds that of
the t conformer in the neutral and monocationic forms. In the
dicationic form the t and g populations are equal. Considering
the twofold occurrence probability of the gauche conformation,
it can be stated that rotamer t is the energetically more stable
rotamer. This phenomenon can be interpreted in steric terms,
since rotamer t contains bulky groups in the remote trans
position. Furthermore, water accessibility to the amino or
ammonium moieties is also favourable in the trans posi-
tion.39 Rotamer t is most populated in the dicationic form, a
consequence of the electrostatic repulsion between the two
positively charged groups. The relatively higher mole fraction
of rotamer g in the neutral and monocationic histamine can be
explained by the lack of cation–cation repulsion, but also by
the stabilising effect of intramolecular hydrogen bonds between
the amino and imidazole groups. There is no significant differ-
ence between rotamer populations in these protonation states,
since the major factors governing the formation of conformers
are similar in these cases.
ϩ
2ϩ
Observed proton
δHis/ppm
δHHis /ppm
δH His /ppm
R
2
Imidazole-2-H
Imidazole-4-H
7.667
6.911
7.723
7.043
8.669
7.404
0.9997
0.9998
libria. Macroscopic protonation constants can be expressed in
terms of the concentrations of macrospecies:
(4)
The observed 1H NMR chemical shifts depend on pH (Fig. 3)
Rotamer-specific basicities
Fig. 2 shows the bulk and rotamer-specific protonation equi-
Fig. 3 Chemical shift–pH functions of imidazole C-2 and C-4 protons
of histamine.
and can be formulated as follows:
ϩ
ϩ
2ϩ
2ϩ
δobs = δHisαHis ϩ δHHis αHHis ϩ δH His αH His
(5)
(6)
2
2
2ϩ
where α values are exemplified by αH His in eqn. (6)
2
From NMR–pH titration curves of the imidazole protons
the macroscopic protonation constants were determined by
non-linear parameter fitting and are in agreement with liter-
ature data.5–7 The macrospecies-specific chemical shifts and
correlation coefficients of the non-linear parameter fitting are
listed in Table 2.
Definition and evaluation of the rotamer-specific basicities is
exemplified below by rotamer t
t ϩ Hϩ
Htϩ
(7)
(8)
Fig. 2 Bulk and rotamer-specific protonation equilibria of histamine.
916
J. Chem. Soc., Perkin Trans. 2, 2002, 914–917

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Table 3 Macroscopic and rotamer-specific protonation constants of
histamine
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log K1 = 10.16
log K2 = 6.35
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log K1t = 10.12
log K1g = 10.18
log K2t = 6.47
log K2g = 6.26
where K1t quantifies the t-rotamer-specific amino basicity of
histamine.
Rotamer concentrations can be expressed with macroscopic
concentrations and rotamer mole fractions:
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[t] = [His]αt
(9)
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[Htϩ] = [HHisϩ]αHt
(10)
ϩ
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Introducing eqns. (9)–(10) into eqn. (8) yields:
(11)
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Logarithmic values of the macroscopic and rotamer-specific
protonation constants are collected in Table 3. Although
macroscopic constants, in principle, cannot be assigned to
specific sites, constants with subscripts 1 and 2 practically
purely characterise the basicity of the amino and imidazole
groups, respectively.
The rotamer-specific amino protonation constants differ
insignificantly from each other and the respective bulk con-
stant. The imidazole protonation exerts a more significant effect
on the rotamer populations. The low log K2g value shows that
uptake of a second proton in the gauche rotamer brings about
breakdown of the monocationic hydrogen bond, and this pro-
cess needs a relatively high hydrogen ion concentration. The
evaluated log K2t value mainly reflects the basicity of the imid-
azole moiety, which is undisturbed by intramolecular hydrogen
bonds, but is certainly influenced by the skeleton-mediated
electron-withdrawing effect of the adjacent ammonium site.
The above statements support our observations on the amino
basicity of rotamers. The higher log K1g value indicates that
the imidazole group promotes the amino protonation by intra-
molecular, inter-moiety hydrogen bond formation in gauche
amino-imidazole positions only.
30 R. J. Sundberg and R. B. Martin, Chem. Rev., 1974, 74, 471.
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Acknowledgements
33 F. Kundell, Ber., 1901, 34, 637.
This work has been supported by the grants MKM FKFP
1126/1997, NjM 447/96, and SE 155/2000.
34 J. W. Conforth and H. T. Huang, J. Chem. Soc., 1948, 1960.
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