Bare histidine-serine models: Implication and impact of hydrogen bonding on nucleophilicity

Article abstract of DOI:10.1002/chem.201301275

A new family of 2-hydroxyalk(en/yn)ylimidazoles has been evaluated as serine-histidine bare dyad models for the ring-opening reaction of L-lacOCA, a cyclic O-carboxyanhydride. These models were selected to unravel the implication of intramolecular hydrogen bonding and to substantiate its influence on the nucleophilicity of the alcohol moiety, as it is suspected to occur in enzyme active sites. Although designed to exclusively facilitate the preliminary step of proton transfer during the studied ring-opening reaction, these minimalistic models depicted a measureable increase in reactivity relative to the isolated fragments. A couple of reliable experimental and theoretical methods have been developed to readily monitor the strength of the intramolecular hydrogen bond in dilute solution. Results show that the folded conformers are the most nucleophilic species because of the intramolecular hydrogen bond. Copyright

Full text of DOI:10.1002/chem.201301275

FULL PAPER
A model family: A new family of 2-
hydroxyimidazoles with various linkers
has been evaluated as serine–histidine
bare dyad models. The presence and
strength of intramolecular hydrogen
bonding has been evidenced and eval-
uated by spectroscopic and computa-
tional methods (see figure).
Hydroxyimidazoles
J. Leclaire,* M. Mazari, Y. Zhang,
C. Bonduelle, O. Thillaye du Boullay,
B. Martin-Vaca, D. Bourissou,*
I. De Riggi, R. Fortrie, F. Fotiadu,*
G. Buono ...................... &&&& — &&&&
Bare Histidine–Serine Models: Impli-
cation and Impact of Hydrogen
Bonding on Nucleophilicity
Marseille–Provence…
…is the European Capital of
Culture in 2013. In this issue two
articles from the Marseille-based
team Chirosciences analyze the
role of imidazole alcohol hydrogen
bonding in the nucleophlicity of
dyad models (see J. Leclaire, D.
Bourissou, F. Fotiadu et al. on
page && ff.) and describe a facile
synthesis and assemblies of tetraal-
kylporphyrins in two and three
dimensions (see Y. Kikkawa, T. S.
Balaban et al. on page && ff.).
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DOI: 10.1002/chem.201301275
Bare Histidine–Serine Models: Implication and Impact of Hydrogen Bonding
on Nucleophilicity
Julien Leclaire,*[a] Messaoud Mazari,[a] Yuan Zhang,[a] Colin Bonduelle,[b]
Olivier Thillaye du Boullay,[b] Blanca Martin-Vaca,[b] Didier Bourissou,*[b]
Innocenzo De Riggi,[a] Rꢀmy Fortrie,[a] Frꢀdꢀric Fotiadu,*[a] and Gꢀrard Buono[a]
Abstract:
A
new family of 2-
sites. Although designed to exclusively
facilitate the preliminary step of proton
transfer during the studied ring-open-
ing reaction, these minimalistic models
depicted a measureable increase in re-
activity relative to the isolated frag-
ments. A couple of reliable experimen-
tal and theoretical methods have been
developed to readily monitor the
strength of the intramolecular hydro-
gen bond in dilute solution. Results
show that the folded conformers are
the most nucleophilic species because
of the intramolecular hydrogen bond.
hydroxyalkACHTUNGTRENNUNG(en/yn)ylimidazoles has
been evaluated as serine–histidine bare
dyad models for the ring-opening reac-
tion of l-lacOCA, a cyclic O-carbox-
yanhydride. These models were select-
ed to unravel the implication of intra-
molecular hydrogen bonding and to
substantiate its influence on the nucle-
ophilicity of the alcohol moiety, as it is
suspected to occur in enzyme active
Keywords: amino acids
functional calculations · hydrogen
bonding NMR spectroscopy
nucleophilicity
· density
·
·
AHCTUNGTRENNUNG
Introduction
The family of serine hydrolases, which is involved in numer-
ous biological processes and widely used in industry,[1] has
been extensively studied over several decades, thus provid-
ing a large amount of structural data. Its active site is char-
acterized by an aspartic acid–histidine–serine (Asp–His–Ser)
triad that undergoes a two-step O-acylation, O-deacylation
process (Scheme 1). As aliphatic alcohols are much less nu-
cleophilic than imidazoles, a basic question concerning
serine hydrolases is how this catalytic triad imparts high nu-
cleophilic reactivity to the serine b-OH group in the first re-
active step (the formation of a covalent tetrahedral adduct
between this residue and the substrate). It is commonly be-
lieved to be achieved by Asp–His and His–Ser intramolecu-
lar hydrogen bonding (IMHB) because it cannot exclusively
be attributed to medium effects in the active-site cleft.
Scheme 1. Proposed mode of action of the Asp–His–Ser catalytic triad of
serine hydrolases/lipases in the presence of a competing hydrogen-bond
donor such as water.
The Asp–His IMHB is in fact supposed to strongly preor-
ganize the imidazole ring in such a position that it prefera-
ꢀ
bly abstracts the SerO H proton, with which it is engaged
in an IMHB, rather than reacting on the substrate.[2] Al-
though numerous experimental observations showed that
this strong hydrogen bond[3] conformationally locks the imi-
dazole ring as soon as the resting state of the enzyme is
empty, direct evidence of the Ser–His interaction, which is a
prerequisite to the catalytic cycle, is still lacking to date. No
X-ray picture of a functional and empty active site (i.e., at a
pH at which His is deprotonated and in the absence of inter-
fering salt) clearly depicts a Ser–His hydrogen bond.[4] The
only compelling, though indirect evidence of a His–Ser hy-
drogen bond comes from liquid-state NMR spectroscopic
studies.[5]
[a] Dr. J. Leclaire, Dr. M. Mazari, Y. Zhang, Dr. I. De Riggi,
Dr. R. Fortrie, Prof. F. Fotiadu, Prof. G. Buono
Centrale Marseille, Aix Marseille Universitꢁ, CNRS
iSm2 UMR 7313, 13397, Marseille (France)
E-mail: julien.leclaire@centrale-marseille.fr
frederic.fotiadu@centrale-marseille.fr
[b] Dr. C. Bonduelle, Dr. O. Thillaye du Boullay, Prof. B. Martin-Vaca,
Dr. D. Bourissou
University of Toulouse, UPS, LHFA
118 Route de Narbonne
31062 Toulouse CEDEX 09 (France)
and CNRS, LHFA UMR 5069
31062 Toulouse (France)
E-mail: dbouriss@chimie.ups-tlse.fr
Whereas the catalytic power of this class of hydrolytic en-
zymes results from a subtle combination of various sophisti-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301275.
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Histidine–Serine Models
FULL PAPER
cated and efficient strategies,[6] a key question remains to be
answered: Although so far unobserved, how might this Ser–
His IMHB exist, be favored by some preorganization, and
lead by itself (i.e., in the absence of any other effect) to a
measureable increase in the nucleophilicity of the alcohol
moiety? Answering this question formally requires one to
extract the dyad from the highly complex protein shell and
study it as a single isolated molecular species.
drogen-bond donors than alcohols, and should therefore
preferentially associate with an imidazole ring as an accept-
or. By using the thermodynamic semiquantitative a,b rank-
ing recently developed by Hunter,[10a] the equilibrium con-
stant for an intermolecular alcohol–imidazole association
should reach at the maximum the modest value of Kinter
=
0.1m.[10b] From this simple analysis, one can conclude that if
the Ser–His IMHB does exist, its intrinsic stability is poor
and the association should clearly be favored by intramolec-
ular effects that are usually gathered into the effective mo-
larity. To confirm this hypothesis, a,w-2-hydroxyimidazoles
of various chain lengths and flexibilities have been synthe-
sized and studied.
So far, only a few studies in physical organic chemistry
have focused on the nucleophilicity of Ser–His bioinspired
dyad[7] models, whereas various His–Asp counterparts have
been synthesized and analyzed.[8] Pioneering work conduct-
ed in the 1980s revealed that the kinetics of acylation/deacy-
lation on 2- and 4-hydroxymethylimidazoles can be modulat-
ed by the susbtituents of the heteroaromatic ring.[9] Whereas
no IMHB was experimentally observed and identified as the
source of the activation of such structures, it was principally
concluded that the imidazole ring acted as a general base.
To address the questions of 1) the impact of preorganiza-
tion on the formation an IMHB within the Ser–His dyad
prior to substrate attack and 2) its potential implication on
the nucleophilicity, we chose to prepare and study a series
of six a,w-2-imidazole alcohols 1–6 as new synthetic mini-
malistic models. The results are reported herein. Spacers of
different lengths and degrees of flexibility have been incor-
porated between the chemically active groups. The nucleo-
philicity of these model dyads has been evaluated in a
model acylation reaction, namely, the ring opening of an O-
carboxyanhydride. The pKa values of compounds 1–6 have
been determined by using a pH meter and their propensity
to adopt folded intramolecularly hydrogen-bonded struc-
tures in solution has been assessed by variable-temperature
1H NMR spectroscopy. In addition, DFT calculations have
been performed to gain more insight into the conformation-
al landscape and to estimate the strength of the intramolec-
ular hydrogen bond.
The ease of intramolecular connection of an a,w-bifunc-
tional structure is known to be strongly influenced by the
number of single bonds in the chain, which determines the
strain of the ring being formed but also the torsional entro-
py lost upon connection.[11] The series explored here was
hence limited to potential five- to seven-membered rings (as
the minimal ring strain corresponds to five or six centers)
with various conformational flexibilities. Compounds 1–6
were prepared in a few steps and good overall yield
A
Scheme 2. 2-Hydroxyalk(en/yn)yl imidazoles 1–6 synthesized and studied
as Ser–His dyad models.
Results and Discussion
tails). The 2-hydroxymethyl derivative 1 was obtained by
direct reduction of the 2-formyl precursor[12a] according to
reported procedures.[12b–e] Selective lithiation of the C2 posi-
tion of N2-sulfamoyl imidazoles, followed by ethylene oxide
ring opening,[13] yielded after deprotection the hydroxyethyl
derivative 2,[14a,b] as previously described. This strategy was
extended to N-protected 2-methylimidazole, thereby provid-
ing a new way to access compound 3.[14c] New dyads 4–6
were obtained through a Wittig reaction between 2-formyli-
midazole and ethyl(triphenylphosphoranylidene)acetate
ylide (for diastereomers 4 and 5) and through Sonogashira
coupling between 2-iodo-1-[2-(trimethylsilyl)ethoxymethyl]
(SEM) imidazole and propargylacetate (for compound 6).
The Wittig coupling procedure developed by McNab et al.[15]
was improved by using tetrahydrofuran instead of pyridine
or benzene as the solvent. This led to a shorter reaction
time (48 h instead of 1 week for complete conversion),
higher yields, and an equimolar mixture of Z/E compounds.
Synthesis of dyad models 1–6: So far, Ser–His dyad models
synthesized to probe cooperative effects between the func-
tional moieties have mostly been limited to hydroxymethyl-
ACHTUNGTRENNUNG
imidazoles.[9] We chose to explore the impact of the number
of rotatable bonds between the primary alcohol and the N-
heterocyclic base on the nucleophilicity of the resulting
structures. The underpinning questions we intended to ad-
dress were the influence of the degree of conformational
preorganization between both groups on the strength of the
potential hydrogen bond between them and the hypothetical
correlation between this noncovalent bond and a kinetic
effect. It is important to note here that, from the point of
view of physical organic chemistry, intermolecular hydrogen
bonding between an alcohol and an imidazole is unlikely to
occur in the presence of many amides (all around the pro-
tein active site) and eventually also in the presence of water
molecules. Indeed, amides and water are slightly better hy-
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J. Leclaire, D. Bourissou, F. Fotiadu et al.
Hydride reduction of the N-protected diastereoisomers[16a]
was preferred over hydrogenation[16b] as it was 100% che-
moselective toward the ester moiety. Although dimethsulfa-
moyl was a suitable protecting group for the synthesis of
compounds 1–5, the Sonogashira coupling proceeded with
higher conversion with SEM, which allowed further orthog-
onal O,N-deprotection reactions.
plays the role of a competing hydrogen-bond donor, an
aprotic polar solvent such as acetonitrile appeared suitable
to potentially observe any nucleophilic behavior of the
dyad. Acetonitrile, like water and amides, although a
weaker hydrogen-bond acceptor than imidazole, should not
allow the formation of an intermolecular imidazole–alcohol
hydrogen bond (DDrG8= +2 kJmolꢀ1).[10b] After 5 min at
1
room temperature, H NMR spectroscopy provided the pro-
Reactivity of 1–6 in a model acylation reaction: To assess
and compare the nucleophilicity of the hydroxylimidazoles
1–6, we studied the ring opening of the O-carboxyanhydride
derived from l-lactic acid (l-lacOCA) 7.[17a] This highly re-
active compound was shown to be an activated equivalent
of lactide towards ring-opening polymerization (ROP). By
using 4-dimethylaminopyridine (DMAP) as catalyst and an
alcohol as initiator, polylactic acid (PLA) of controlled
molar mass and narrow polydispersity was prepared under
mild conditions.[17a,b] The mechanism of the elementary ring-
opening step has been thoroughly studied computationally,
and the pathway that involves basic activation of the alcohol
was found to be favored over the nucleophilic activation of
the O-carboxyanhydride.[17c] Note that lipase-mimicking
compounds have also recently started to be explored as cat-
alysts for the ROP of lactide.[17d]
portion of products, which remained subsequently constant,
thus indicating that the conversion of this kinetically con-
trolled reaction was complete. Products that resulted from
the attack of alcohol moieties on the anhydride of l-
lacOCA were exclusively obtained. Integration of the CH2O
signals provided the proportion of products 8–13 relative to
methyl-l-lactate 14 (Table 1).
Table 1. Distribution of products 8–13 relative to methyl-l-lactate 14 re-
sulting from the nucleophilic attack on l-lacOCA 7 (c7 =0.1m) in acetoni-
trile; kinetic effective molarity (EM); and pKa of the dyad mimics 1–5
measured at 0.1–0.001m concentrations in 1m aqueous KCl at 300 K.
1
2
3
4
5
6
LacOIm [%]
8–13
15
40
10
50 (93)[a]
0
0
EM=kI/kM
pKa
3
7.33
8
7.00
2
7.07
10.1
4.36
–
5.71
–
4.80
Compounds 1–6 combine two such moieties, a primary al-
cohol and a basic imidazole. They were first evaluated indi-
vidually in the ring opening of 7 in the presence of methanol
as a competitor (Scheme 3). A control experiment showed
[a] c7 =0.001m.
These preliminary experiments conducted independently
on compounds 1–6 provided a first classification of their rel-
ative nucleophilicity with respect to methanol (Table 1). De-
spite the strong excess amount of methanol used, dyad ad-
ducts 8–13 were obtained in significant amounts. Decreasing
the proportion of methanol to 10 equiv yielded these com-
pounds as major products that could subsequently be puri-
fied by column chromatography and fully characterized (see
the Supporting Information). Within the saturated series 1–
3, hydroxyethylimidazole 2 appeared as the most active
compound, potentially involving
a six-membered ring
IMHB during the nucleophilic activation process. In the C3
series 3–5, a marked difference in reactivity was observed
between Z and E unsaturated isomers, the former being the
most reactive (five times more than the saturated analogue
3), whereas the latter, similarly to the alkynyl model 6, did
not lead to any detectable amount of corresponding lactate
adduct (Table 1). Although 5 and 6 do not benefit from any
intramolecular activation, because of conformational restric-
tions, their total absence of reactivity in the presence of
methanol provides an insight into the detrimental electronic
impact of the bridging unsaturation on the nucleophilicity of
the terminal alcohol moiety. The propenenyl and propargyl
alcohol residues are indeed expected to be intrinsically less
nucleophilic than methanol.[18]
Scheme 3. Putative reactive complexes in rapid interconversion leading
to l-lactate adducts 8–13.
that no reaction occurs between methanol and l-lacOCA 7
in the absence of a base. To avoid any polymerization and
limit the reaction to a single coupling, a strong excess
amount of methanol (90 equiv, which in fact represents a
40:60 MeOH/CH3CN mixture) was used relative to 7 (c7 =
0.1m). Comparatively, a slight excess amount of the dyad
model 1–6 (1.5 equiv) was introduced. As methanol already
To confirm this preliminary classification, competition ex-
periments that involved two hydroxyimidazoles and an
excess amount of methanol were conducted under identical
conditions (see the Supporting Information). This second set
of data confirmed the higher activity of 2 with respect to 1
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Histidine–Serine Models
FULL PAPER
and 3, and of 4 with respect to 3, 5, and 6. In the saturated
series, the variation in the number of carbon atoms proved
to have a crucial impact on the reactivity. In the second
series, although the length of the aliphatic chain linking the
base to the nucleophile is fixed, the accessible conformation-
al space notably differs, thus leading to an even greater dis-
crimination in terms of activity. Direct confrontation be-
tween the most active derivatives 2 and 4 yielded a balanced
mixture of corresponding adducts (40:40) in full agreement
with individual measurements.[19]
In terms of reactive pathways (Scheme 3), the trimolecu-
lar mixture of reactants (compounds 1–6, methanol, and l-
LacOCA) can either react through an intramolecular activa-
tion of the dyad (rate constant kI) to lead to 8–13 or through
the intermolecular activation of methanol by the imidazole
ring of the dyad (rate constant kM) to lead to 14. The first
pathway involves a bimolecular reactive complex with which
methanol might hypothetically interact as a hydrogen-bond
celeration observed during the cyclization of preorganized
diester systems designed by Bruice and co-workers.[22c] In
practice, methanol must indeed be used as a cosolvent to be
kinetically competitive with the dyad models. This translates
into a reduction of the activation energy of 1.7–5.7 kJmolꢀ1
with a barrier of 60–70 kJmolꢀ1. Such a value underpins a
noticeable effect during the first step (the activation/depro-
tonation of the alcohol) of a complex molecular process of
addition/elimination that involves the breakage and forma-
tion of several covalent bonds. Within the dyad series, the
difference in the energy of activation between 3 and 4 (the
least and most reactive dyads) of 5.1 kJmolꢀ1 provides a
quantitative estimate for the advantage of freezing out a
rotor during the formation of an intermolecular noncovalent
bond, which is in agreement with earlier reports.[23]
Physical indicators of nucleophilic activation: The pKa
values of the imidazolium salts of compounds 1–6 were
measured by half-protonation of the conjugated neutral
form in 1m KCl aqueous solutions in a concentration range
of 0.1 to 0.001 molLꢀ1, with each point being run in tripli-
cate. Alkenyl and alkynyl derivatives 4, 5, and 6 displayed
lower values than the saturated series 1–3, thereby revealing
the influence of the connecting unsaturated bond on the ba-
sicity of the heterocyclic nitrogen center, although the lone
pair and the carbon–carbon double bond are in orthogonal
orbitals. Within diastereoisomers, the configuration of the
carbon–carbon double bond has a dramatic impact on the
acidity of the imidazolium ring: the pKa difference between
the conjugated acids of Z and E diastereoisomers 4 and 5
reaches 1.4 units, the former being the most acidic. For the
saturated series 1–3, an evolution of moderate amplitude is
observed (0.33 pKa units). The C2-bridged compound 2 is
the least basic, in contrast with early measurements on 2-hy-
droxyalkylpyridines, the basicity of which was reported to
decrease with chain length.[24]
ꢀ
acceptor through an intermolecular O···H N bond, whereas
the second route relies on a trimolecular reactive complex
in which methanol acts as donor and the dyad might hypo-
ꢀ
thetically be engaged in an intramolecular N H···O bond.
The two reactive complexes might interconvert through a
hydrogen-bond rescrambling process with an equilibrium
inter
ðOꢀHꢂꢂꢂNÞ
intra
ðOꢂꢂꢂHꢀNÞ
intra
ðOꢀHꢂꢂꢂNÞ
inter
ðOꢂꢂꢂHꢀNÞ
constant K=[K
ꢂK
]/[K
ꢂK
] to
reach values of 6.3 and 25 for 2 and 4, respectively. This
equilibrium roughly evaluates the difference in between
ꢀ
ꢀ
intra- and intermolecular N H···O and O H···N hydrogen
bonding (from 4.5 to 8 kJmolꢀ1 for 2 and 4, respectively).[20]
The difference in molecularity between the two pathways in-
volved in this model could be experimentally confirmed by
using a fixed stoichiometry between the three molecular
partners (4, MeOH, and l-lacOCA) while decreasing the
overall concentration (c7 =0.1 to 0.001 molLꢀ1) in the reac-
tive aliquots (Table 1). Gas-phase chromatography was used
on this range of concentrations to monitor the distribution
1
of adducts 11 and 14 in complement to H NMR spectrosco-
At this stage, it is interesting to compare the pKa values
of the imidazolium salts with the nucleophilicity of their
conjugate base 1–6, as quantified by the kinetic constants of
the l-lacOCA ring opening. In his early work on hydroxy-
methylimidazoles, Brown et al. found a strong correlation
between nucleophilicity and basicity; the more acidic the
imidazolium salt was, the more reactive the conjugated base
in model acylation reactions.[25] For this hydroxymethyl
series, increasing the electronic density on the heteroaro-
matic ring increases both the intrinsic hydrogen-bond donor
character of the imidazole and the basicity, whereas the
degree of preorganization (and hence the effective molarity)
remains constant. In contrast, in both the alkyl- and alkenyl-
bridged series 1–3 and 4–6, the most reactive compound to-
wards l-lacOCA was found to be the least basic (Table 1).
In this collection, the nature of the spacer affects the elec-
tronic density, and therefore the intrinsic donor character
and the basicity of the imidazole to a certain extent, but it
also strongly and independently affects the effective molari-
ty by spatial preorganization. The decorrelation between in-
trinsic hydrogen-bonding character and degree of preorgani-
py. The amount of methyl-l-lactate 26 was precisely calibrat-
ed and monitored in the various reaction mixtures. It yield-
ed, in agreement with the model, a linear distribution of the
product ratio [14]/[11] with increasing concentrations.[21] For
millimolar concentrations, at the lowest detection limit of
methyl-l-lactate by the chromatographic method, adduct 11
was obtained in 93% yield. From the product distribution
previously discussed, the corresponding ratio of kinetic con-
stants kI/kM could easily be extracted. This rate corresponds
to the kinetic effective molarity (kEM), an indicator of the
relative intrinsic reactivity between the intra- and intermo-
lecularly activated reactive states (see Table 1 and the Sup-
porting Information). This indicator of the benefit of the in-
tramolecular preorganization on the reactivity, which repre-
sents the hypothetical methanol concentration required for
the intermolecular process to compete with the intramolecu-
lar one, varied between 2 and 10m within the dyad series. It
is one order of magnitude higher than the acceleration in-
duced by supramolecular precomplexation of reactive part-
ners,[22a,b] but several orders of magnitude lower than the ac-
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J. Leclaire, D. Bourissou, F. Fotiadu et al.
zation hence reasonably explains the absence of general cor-
relation between the Brønsted basicity and nucleophilicity
evaluated experimentally.
In the context of hydrogen-bonded structures, tempera-
ture coefficients dd/dT provide a finer level of analysis than
the chemical shift, thus allowing one to differentiate be-
tween protons engaged or not in such interactions and also
to differentiate those interacting with the solvent from those
protected from the bulk solution and engaged in strong in-
tramolecular bonds. As revealed in Table 2, while the chemi-
1
Direct observation of the IMHB by H NMR spectroscopy
in highly diluted conditions: As mentioned earlier, the only
experimental evidence of the IMHB in serine hydrolases to
date is the downfield shift of the Ne2 His signal in solution-
state 15N NMR spectroscopy upon the chemical deactivation
of the proximal b-OH catalytic group (Scheme 1), as the
proton involved in the IMHB is replaced by a neutral sub-
stituent. This selective neighboring effect of the chemical
mutation was therefore reported to demonstrate the exis-
Table 2. Temperature coefficients [ppbKꢀ1] between 300 and 215 K for
compounds 1–6.
d[d(OH)]/dT
d[d(CH2a)]/dT
d[d(NH)]/dT
1
2
3
4
5
6
ꢀ8.18
ꢀ7.75
ꢀ8.43
ꢀ1.43
ꢀ8.16
ꢀ6.82
0.45
1.63
0
0
0.34
0
ꢀ7.50
ꢀ7.38
ꢀ5.43
ꢀ6.57
ꢀ4.46
ꢀ5.87
ꢀ
tence of a N···H O-type hydrogen bond between His and
Ser in the resting state in solution.[26,27]
Direct experimental observations of O H···N(sp ) IMHBs
remain extremely scarce, and comparatively, evidence for
2
ꢀ
2
ꢀ
N H···O(sp ) systems is relatively abundant. Still, NMR
spectroscopy stands as a method of choice for detecting hy-
drogen bonds[28–30]: in nonprotic and relatively dry media,
NH and OH protons can easily be observed, and they usual-
ly resonate at higher chemical shifts when hydrogen-
bonded.
cal shift of the non-hydrogen-bonded protons such as
CH2OH slightly shifts downfield with increasing tempera-
ture, hydrogen-bonded ones such as NH and OH display a
strong upfield variation.[33] In fact, within hydrogen-bonded
protons, the pivotal value of the temperature coefficient
that discriminates between intermolecularly solvent-bonded
and intramolecularly solute-bonded protons was set at
around ꢀ4.5 ppbKꢀ1 for amides in peptides[34] and around
ꢀ3 ppbKꢀ1 for hydroxyl in saccharides.[35] Downfield shifts,
smaller temperature coefficients, slower rates of exchange,
and lower vicinal coupling constants (3J(OH,CHn)) are usu-
ally indicative of strong hydrogen bonds, four factors that
were met only with compound 4. The OH temperature coef-
ficient of this active dyad model was superior to
ꢀ1.5 ppbKꢀ1, whereas the other compounds displayed an
average value of ꢀ8 ppbKꢀ1. The strong reactivity and low
pKa value displayed by 4 appeared to indicate a tight IMHB
at 300 K, which, according to the series of spectra recorded,
was reinforced at lower temperatures. In terms of statistics,
the intramolecular hydrogen-bonded conformer should in
fact strongly dominate the
In the present study, 2-hydroxyalk(en)ylimidazoles 1–6
were dissolved in [D7]DMF at 1 mm concentration.[31] Previ-
ous studies reported that self-associations of a,w-hydroxyal-
kylpyridines were characterized by molar affinities in apolar
solution.[32] Therefore, noncovalent oligomeric associations
between hydroxyimidazoles should be ruled out under the
present analytical conditions. To further mimic the solvation
state of the microenvironment of hydrolytic active sites, an
equivalent of water was introduced as a potential intermo-
lecular hydrogen-bond competitor. Spectra of 1–6 displayed
large signals for OH and NH protons at room temperature,
with the former sharpening into a well-resolved triplet
t(3J(OH,CH2) coupling) near the freezing point of DMF
(Figure 1). As expected, both types of nuclei were more de-
shielded at low temperatures at which hydrogen-bonded and
stabilized conformers should be more populated.
global population at this tem-
perature and below. This pic-
ture was confirmed by theoreti-
cal calculations (see below).
In silico conformational study
of dyad models 2 and 4: The
conformational populations of
the most nucleophilic dyads 2
and 4 of each series (saturated/
unsaturated) were estimated by
DFT calculations by using four
different methods and including
solvent correction (see the Sup-
porting Information). Coherent
Figure 1. Variable-temperature 1H NMR (400 MHz) spectra of 1 (1.0 mm) in [D7]DMF): a) 300, b) 280, c) 235,
d) 225, and e) 215 K.
results were obtained with the
different methods, and the dis-
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Histidine–Serine Models
FULL PAPER
cussion will be focused here on the results obtained at the
M06-2X/6-31++G**+SMD level of theory, which is particu-
larly relevant for hydrogen-bonded systems.[36] The results
and even more its variation with temperature, should be the
most relevant indicator. And indeed, it appears that CE,
which should be reasonably related to the averaged chemi-
cal shift,[37] undergoes a much stronger variation for 2 than
for 4 on the temperature scale experimentally explored by
NMR spectroscopy.
In light of the NMR spectroscopic data, these calculations
seem to reveal a conformational landscape dominated by a
folded species 4c up to room temperature. This tendency is
underpinned by a slightly stronger IMHB for 4 than for 2.
In both cases, the intramolecular binding constant should be
about 5–10 kJmolꢀ1, which matches the average value
(6 kJmolꢀ1) of the entropic penalty that has to be paid to
immobilize molecular partners through noncovalent inter-
molecular interaction. These orders of magnitude confirm
1
were compared to the variable-temperature H NMR meas-
urements to determine the influence of the conformational
distribution on the averaged magnetic signals. The propor-
tion of the activated conformers and the level of their calcu-
lated molecular orbitals were additionally related to the re-
activity evaluated experimentally to determine the potential
correlation between the degree of preorganization in the re-
active states and the reactivity.
The results summarized in Figure 2 indicate that 2 and 4
do not fundamentally differ in terms of conformational land-
scape at 298 K, which seems at first sight to disagree with
the NMR spectroscopic measurements. In fact, at room tem-
perature, around 85% (respectively 75%) of dyad 4 (respec-
ꢀ
that an N···H O hydrogen bond might only occur intramo-
ꢀ
tively 2) adopts the O H···N intramolecularly bonded con-
lecularly in polar and protic media and is modulated
through the conformational preorganization provided by the
spacing unit. In the present case, thermodynamic effective
molarities can be estimated to be around 102 and 4ꢂ102 for
2 and 4, respectively. In terms of kinetics, DFT calculations
including solvent correction conducted on intramolecularly
bound and unbound conformers of 2 and 4 indicate that
within the framework of a natural bond orbital (NBO) anal-
ysis, the oxygen lone pair that is involved in the ring-open-
ing reaction of l-lacOCA is significantly higher in energy in
the bound than the unbound conformer. Concomitantly, the
nitrogen lone pair is significantly stabilized when the IMHB
formation, the distribution over the minor species being
broader for 2 than for 4, however. These latter species,
ꢀ
being either unfolded or displaying the reversed N H···O
IMHB, are destabilized by 5–7 kJmolꢀ1 for 2 (and 6–
13 kJmolꢀ1 for 4) with respect to the ground-state structure,
which provides a rough estimation of the intramolecular
binding constant and of the hydrogen-bond strength for
both dyad models. Considering the nature of the experimen-
tal parameter that allows for the detection of the IMHB by
NMR spectroscopy, the conformational excess (CE) be-
ꢀ
tween the N H···O bonded species and the other species,
Figure 2. Analysis of the conformational population at thermodynamic equilibrium of the most active dyad models 4 (gray) and 2 (black) at 298 and
215 K. In both cases, the SMD/M06-2X/6-31++G** level of theory has been used (see text for a full description of calculations). Bold numbers stand for
conformer labels and fractions. Central frame: corresponding conformational excess (CE) with respect to the temperature at the same interval for com-
pound 2 (black) and 4 (gray).
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J. Leclaire, D. Bourissou, F. Fotiadu et al.
is present. As a consequence, the nucleophilicity of the hy-
droxyl group is expected to be significantly elevated by the
IMHB in the bonded conformation.
Experimental Section
Variable-temperature NMR spectroscopic measurements on activated al-
cohols 1–6: NMR spectra at variable temperatures were recorded using a
Bruker AVANCE DPX 400 MHz spectrometer, equipped with a 5 mm
multinuclear reverse Z-gradient probe and using [D7]DMF as the solvent.
The temperature of the probe was calibrated by the methanol standard
method, and a delay of 600 s was used before recording the NMR spectra
at each temperature. The operating frequency was 400.13 MHz for 1H.
Each spectrum was run with a relaxation delay of 2 s, a 308 pulse, a time
domain size of 64 K, and a real transform size of 32 K. Each NMR spec-
troscopic sample was prepared by using a new 5 mm tube capped with a
septum and that contained a small amount of freshly activated 3 ꢃ mo-
lecular sieves (to keep the medium as dry as possible during the overall
NMR spectroscopy experiment) under an inert atmosphere. Then a solu-
tion of compound 1–5 (ꢁ1 mg) in dry [D7]DMF (600 mL; ꢃ99.5 % D,
(HDO+D2O) <0.05%) was transferred with a syringe through the
septum.
Still, whereas the strength of the IMHB and the propor-
tion of the folded conformer differs noticeably between 2
and 4, the two dyad models display similar reactivity toward
l-LacOCA. This observation tends to indicate that, whereas
the IMHB must indeed exist to generate a productive reac-
tive pathway, the increase in nucleophilicity is not correlated
to its strength in the reactive state. This is in agreement with
a Curtin–Hammet-type model in which intra- and intermo-
lecularly bound species are rapidly interconverting, whereas
the rate-limiting step remains as the addition/elimination
processes (Scheme 3).
General procedure for the ring-opening of 7 by a single dyad model 1–6
in the presence of methanol NMR spectroscopic monitoring: In a typical
experiment, a solution of l-lacOCA in CD3CN (20 mg; 0.17 mmol in
2 mL) was added to a solution of 1–6 in CD3OD (0.26 mmol in 0.63 mL).
The mixture was stirred at room temperature for 5 min and then ana-
Conclusion
2-Hydroxyalk(en)ylimidazoles, which are easily accessible
synthetically, have proven to be reasonable serine–histidine
dyad models for detecting the postulated OH···N IMHB in
solution and for evaluating its degree of implication on the
nucleophilicity of the alcohol moiety. In fact, while tested as
nucleophiles for the ring-opening reaction of l-lacOCA,
these minimalistic models displayed higher nucleophilicity
than their intermolecularly activated counterparts. This
result points out that in the absence of additional physical
effects that might be involved within enzyme active sites, a
Ser–His-like IMHB substantially activates O-nucleophilicity,
a phenomenon that potentially contributes to the enzymatic
activity. This noncovalent bond clearly inherently facilitates
the rate-limiting[17c] proton transfer but should be ineffective
in accelerating the creation of the covalent bond between
reactive partners. It is therefore remarkable that the result-
ing impact, although it remains modest and partly related to
a favorable molecularity effect, is experimentally measure-
1
lyzed by H NMR spectroscopy.
Computational methods: Conformational studies were achieved with the
Gaussian 03[38] software suite. Full scans of the rotational degrees of free-
dom of the alkyl chains were first performed at the AM1 level of theory,
with 158 steps being used for dihedral angles. All local minima of the re-
sulting potential-energy surface were subsequently fully optimized at the
B3LYP/6-31G(d,p), B3LYP/6-31++G(d,p), B3LYP/6-31++G(d,p), and
M06-2X/6-31++G levels of theory, with the nature of the stationary
points being systematically tested through vibrational frequency calcula-
tions. A solvent was introduced as single-point electronic energy correc-
tions using gas-phase optimized geometries and the C-PCM or SMD con-
tinuum model[39] (solvent: DMF,[40] radii: UAKS, tesserae: 0.2). Finally,
Gibbs free energies were estimated by using a homemade Python script
with the usual ideal gas/rigid rotator/harmonic oscillator approximation
and the ideal solute thermodynamic reference (infinitely diluted solute
with a reference concentration of 1 molLꢀ1).
X-ray structural analysis: Detailed crystal structures, cell parameters, and
R values for 2 and 9 are reported in the Supporting Information.
CCDC-932593 (2) and 932494 (9) contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif.
ACHTUNGTRENNUNGable. Whereas the basicity of the imidazole ring has often
been evoked as an indicator of the extent of the nucleophilic
activation, it has presently been proven not to correlate with
the reactivity. In this series, variation in the length and flexi-
bility of the spacers strongly biased the preference between
intra- and intermolecular proton abstraction, thereby alter-
ing the pKa and the O-nucleophilicity of the entities by con-
struction. Concerning the IMBH, a couple of simple experi-
mental and theoretical methods have been applied to easily
detect the presence of IMHB in dilute solution in the pres-
ence of polar and protic competitors. The resulting data col-
lected led to the conclusions that the intramolecular hydro-
gen-bonded species might only be detected when strongly
dominating the conformational landscape, and that the spe-
cies arises from the conformational preorganization between
both moieties (herein provided by the spacing unit but pre-
sumably exerted by the Asp–His interaction in hydrolases)
rather than intrinsic stability. In fact, it clearly appears that
this productive geometry that involves a robust internal hy-
drogen bond provides a significant increase in alcohol nu-
Acknowledgements
Financial support from the French MENRT for M.M. and the Chinese
CSC for Y.Z. is gratefully acknowledged. N. Saffon (Institut de Chimie
de Toulouse) is acknowledged for the X-ray diffraction structures.
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Histidine–Serine Models
FULL PAPER
that the Ser–His dyad is less sequestered to solvent than the His–
Asp counterpart. In the solid phase, NMR spectroscopic studies (see
reference [6]) recently confirmed what X-ray snapshots (see refer-
ence [7]) seemed to point out: the Ser–His intramolecularly bonded
microscopic state does not seem to be sufficiently populated to be
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strates. Bachovchin recently addressed this question by carrying out
equivalent 15N NMR spectroscopic studies on enzyme crystals and
found that there was no His–Ser hydrogen bond in the crystal (see
reference [3]). In fact, as crystals were routinely made in high
Li2SO4 solutions, a sulfate ion was bound in the active site between
His and Ser, thereby raising the pKa of His such that it is protonated
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[7] Qualitatively, the fundamental mechanism of enhancing the nucleo-
philic power of any given oxygen atom is to enhance the electron
density of the nucleophilic lone pair. This leads to two molecular
strategies of generating increased electron density in the reactant
state by altering the coordination and/or bonding of the nucleophile.
The first one is desolvation, or more precisely the stripping of hy-
drogen-bond donors to the nucleophilic lone pair. The second one
consists of coordinating the alcohol proton (i.e., hydrogen bonding
to a general base that might ultimately lead to its ionization). In ad-
dition to these two mechanisms, an electric field generated at the
active site can polarize a bond to the nucleophile, thereby enhancing
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philicity of the alcohol moiety. In fact, measurements on primary
amines have revealed the following order of nucleophilicity:
ꢀ
ꢄ
MeNH2 >H2C=CH CH2NH2 ꢁEtNH2 >CH CCH2NH2. For more
details, see: F. Brotzel, Y. C. Chu, H. Mayr, J. Org. Chem. 2007, 72,
3679–3688.
[19] The remaining 20% correspond to higher oligomers. On the basis of
k values, the theoretical ratio is 36:44.
[20] Whereas the intermolecular constant can be roughly evaluated by
using Hunterꢄs model detailed in reference [10a], the ratio of intra-
molecular binding constants was deduced from the calculations of
the Gibbs enthalpy of the corresponding bonded states (see below
and the Supporting Information).
[21] For the trimolecular scenario 4+MeOH+7: d[LacOMe]/c0dt=
kM[ImOH]/c0[MeOH]/c0[LacOCA]/c0, whereas the bimolecular sce-
nario 4+7 follows: d[ImOMe]/c0dt=kI[ImOH]/c0[LacOCA]/c0, with
[MeOH] being approximately constant and roughly equal to
90c7(t=0), the resulting rate gives: d[LacOMe]/d[LacOMe]=kM/
kI [MeOH]ꢂ90c7(t=0)/c0 =k’c7(t=0). For more details, see the Sup-
porting Information.
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IMHB is clearly geometrically inaccessible. Such a tendency could
therefore not be directly attributed exclusively to the IMHB
strength because it is subjected to electronic parameters such as a
through-bond inductive effect of the OH group.
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,
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anilide) and pKa value. See reference [30d,e].
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[27] The similar magnitudes of the hydrogen-bond perturbations of N1
and N3 chemical shifts as donors and acceptors indicate at least
qualitatively that the strength of the Asp–His and His–Ser hydrogen
bonds are not very different. For more details, see reference [4].
[28] Although infrared spectroscopy helps in differentiating free, inter-,
and intramolecularly bonded OH groups, establishing a strict hierar-
chy between compounds on the basis of the strength of their IMHB
remains tedious. All the more as one must operate under sufficiently
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ꢀ
[37] Unfolded states and to a lesser extent the N H···O folded state
should display a similar chemical shift for the hydroxyl proton,
which is invariably engaged in intermolecular hydrogen bonding
with the solvent or with water. In contrast, such an exchange is re-
stricted in the intramolecularly bonded species, and the magnetic en-
vironment of the hydroxyl proton is strongly influenced by the imi-
dazole donor.
[30] Such a typical low-field signal was also observed in cis-urocanic de-
rivatives designed as natural and simple His–Asp dyad mimics that
involve a direct covalent linkage of the side-chain functionalities
through a rigid seven-membered hydrogen-bonded ring. See refer-
ence [2].
[31] This solvent appeared to gather a relevant set of physical properties
because it was a hydrogen-bond acceptor and displayed a slightly
higher polarity than the local microenvironment that surrounded
the catalytic residues. For a reference on the local polarity within
the active site of hydrolases, see: a) E. L. Mertz, L. I. Krishtalik,
Proc. Natl. Acad. Sci. USA 2000, 97, 2081–2086; b) L. I. Krishtalik,
Chem. Phys. 2005, 319, 316–329.
[32] NMR spectroscopic studies so far have focused on the resonance
and coupling patterns of the nitrogen acceptor and, less frequently,
of neighboring carbon atoms (see reference [30]). The atoms engag-
ed in the noncovalent bond and, to a lesser extent, the adjacent
carbon atoms can be used as magnetic probes, with hydrogen bond-
[38] Gaussian 03, Revision D.02, M. J. Frisch, G. W. Trucks, H. B. Schle-
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15
13
1
ꢀ
ing influencing their chemical shifts (d N, d( CaHn), d(Ca Hn))
3
1
1
and coupling constants (1J(15N,1H), J( H Ca,N··· H), 1J(13Ca,1H))
with the shared proton. Multidimensional experiments combined
with long acquisition time or isotopically labeled substrates are gen-
erally required when operating under dilute conditions to minimize
intermolecular effects.
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ꢀ
[33] The amplitude of this variation depends, on the one hand, on factors
that are constant for 1–5 such as the nature of the solvent and the
Received: April 4, 2013
Published online: && &&, 0000
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