Elusive Dehydroalanine Derivatives with Enhanced Reactivity

Article abstract of DOI:10.1002/cbic.201800758

For the first time, a simple methodology for the chemical synthesis and use of highly reactive 4-methylenoxazol-5(4H)-ones from serine is presented. These dehydroalanine derivatives, which resemble the natural 4-methylidenimidazole-5-one (MIO) cofactor pr

Full text of DOI:10.1002/cbic.201800758

Accepted Article
Title: Elusive Dehydroalanine Derivatives with Enhanced Reactivity
Authors: Carlos Aydillo, Nuria Mazo, Claudio D. Navo, and Gonzalo
Jiménez-Osés
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10.1002/cbic.201800758
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Elusive Dehydroalanine Derivatives with Enhanced Reactivity
Carlos Aydillo+,[a,b,c] Nuria Mazo+,[a] Claudio D. Navo+,[a] and Gonzalo Jiménez-Osés*[a,d,e]
Abstract: For the first time, a simple methodology for the chemical
synthesis and utilization of highly reactive 4-methylen-oxazol-5(4H)-
ones from serine is presented. These dehydroalanine derivatives,
which resemble the natural 4-methylideneimidazole-5-one (MIO)
cofactor present in lyases and aminomutases, undergo rapid
reaction with carbon nucleophiles such as silyl enol ethers, and
cycloaddition reactions with diazo compounds and reactive dienes
under very mild conditions and in the absence of metal catalysts and
ring-strain activation, offering potential for bioconjugation.
On the other hand, naturally occurring Dha and Dhb have been
functionalized through S-, N- and C-Michael reactions, but
natural reactions involving O-nucleophiles have not been
discovered yet.[9] Hence, the incorporation in peptides and
proteins of a highly reactive ,-dehydroamino acid derivative
able to undergo conjugate additions with weak nucleophiles
such as carbohydrates, which would directly lead to O-
glycopeptides and O-glycoproteins through site-specific
chemical PTM, is still a major challenge in chemical biology.
In our continuous search for ,-dehydroamino acid scaffolds
with improved reactivity, the natural 4-methyliden-imidazole-5-
one (MIO) protein cofactor drew our attention.[10] MIO is
generated as a PTM from the Ala-Ser-Gly triad in ammonia
lyases and aminomutases (Figure 1a,b) and it is responsible for
their activity towards amino acids. The amino groups of aromatic
-amino acids are N-alkylated through an aza-Michael addition
reaction with MIO, which promotes -elimination to give
cinnamic acid derivatives (in lyases) and subsequent
isomerization to -amino acids (in aminomutases). The
structures of the chromophores of the green fluorescent and
related proteins (GFP) are very related to that of MIO,[11] were
the central serine of the triad is replaced by an aromatic residue
such as tyrosine in GFP, leading to stable 4-arylidene-imidazole-
5-ones.
To the best of our knowledge, discrete 4-methylidene-imidazole-
5-ones have not been prepared or isolated outside the protein
context of the MIO cofactor, likely due to their very high reactivity
towards nucleophiles compared to other ,-dehydroamino acid
derivatives. In an attempt to chemically synthetize the MIO
scaffold under physiological conditions, we synthesised the
minimal natural sequence leading to cyclization in lyases and
aminomutases, namely Ac-Ala-Ser-Gly-NH2 (Figure 1c). Solid-
phase peptide synthesis (SPPS) was used to obtain the linear
tripeptide with the C and N termini capped as amides in good
yield (see Supporting Information). The desired spontaneous
cyclization of this triad through intramolecular aminal formation
followed by two consecutive dehydrations, could not be
observed either by 1H NMR spectroscopy or MS spectrometry
after prolonged heating at 50 ºC in pH 8.0 PBS buffer.
Chemical modification of proteins is a very active field of
research in current chemical biology.[1] Such post-translational
modification (PTM) of proteins requires site-selective reactions
with high chemoselectivity.[2] Along these lines, ,-unsaturated
amino acids are of special interest, since they constitute a
modular platform for site-selective PTM,[3] mainly through 1,4-
conjugate addition of thiols such as those found in cysteine
sidechains.[4] However, controlling the stereoselectivity in
reactions involving ,-dehydroamino acids and peptides still
represents a challenge for chemists. In this context, we have
reported the synthesis of chiral dehydroalanine (Dha) and
dehydrobutyirine (Dhb) building blocks and their application in
the asymmetric synthesis of lanthionine and -methyllanthionine
derivatives.[5] Our first-generation chiral Dha scaffold was a
versatile Michael acceptor towards nucleophilic thiols such as
protected 1-thiocarbohydrates.[6] More recently, we developed
an improved version of these chiral Dha/Dhb through
lactonization of the first-generation scaffolds, yielding chiral
bicyclic structures with reduced conformational flexibility and
superior reactivity and diastereoinducing properties with thiols as
nucleophiles.[7] This methodology allowed synthesizing cell-
penetrating peptides containing fluorescent D-cysteines.[8]
Unfortunately, such Dha/Dhb scaffolds were unsuitable for
introducing any other nucleophiles besides thiols, due to their
limited reactivity.
Aiming to facilitate cyclization by bringing the reacting fragments
closer, the stapled peptides Ac-Cys-Asp-Ser-Gly-Cys-NH2 and
Ac-Cys-Lys-Ser-Gly-Cys-NH2 (Figure 1d) were likewise
synthesised by performing an oxidative cleavage from the resin
to form disulfide bonds between the two C- and N-terminal
cysteines (see Supporting Information). The native Ala residue
was mutated to Asp and Lys to solubilize the resulting peptides
in water. These peptides were also heated at 50 ºC in pH 8.0
PBS buffer for many days, but no significant changes were
detected by 1H NMR or MS analyses. These experiments
proved that MIO scaffolds are very difficult to obtain in vitro in
the absence of the protein scaffold of the corresponding enzyme,
which appears to promote cyclization by imposing severe
confinement constraints.[12]
[a]
[b]
Dr. G. Jiménez-Osés, Dr. C. Aydillo, Dr. C. D. Navo, N. Mazo
Departamento de Química.
Universidad de La Rioja.
Madre de Dios, 53, 26006 Logroño (Spain).
E-mail: gonzalo.jimenez@unirioja.es
Dr. C. Aydillo
Department of Pharmaceutical Technology and Chemistry, Faculty
of Pharmacy and Nutrition
University of Navarra, Pamplona (Spain)
Dr. C. Aydillo
Instituto de Investigación Sanitaria de Navarra (IdiSNA)
Irunlarrea 3, E-31008 Pamplona, Spain
Dr. G. Jiménez-Osés
CIC bioGUNE, Bizkaia Technology Park, Building 801A, 48170
Derio (Spain)
Dr. G. Jiménez-Osés
[c]
[d]
[e]
[+]
Ikerbasque, Basque Foundation for Science, Maria Diaz de Haro 13,
48009 Bilbao (Spain).
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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corresponding to a polymeric material. The same results were
obtained with N,N’-dicyclohexylcarbodiimide (DCC) and N,N’-
diisopropylcarbodiimide (DIC) as carboxylic acid activators.
Careful monitoring of the reaction between 1 and DIC in CDCl3
by 1H NMR (Figure 1) showed the fast disappearance of the
starting material signals, and the rise of two narrow doublets in
the 6.00–6.20 ppm region associated to methylene protons,[14]
reaching a maximum conversion of 90% in 15 min. Thus, it was
clear that 4-methylen-2-phenyloxazol-5(4H)-one 2 (abbreviated
as MPO) forms immediately via DIC-promoted cyclization and
subsequent β-elimination of benzoic acid, although this
compound is too reactive to be isolated by a conventional
workup. As expected, the lifetime of 2 depends on the solvent
used in the reaction and the concentration of the sample. In non-
polar solvents such as chloroform, compound
2 can be
preserved in solution for at least 3 h at concentrations up to 190
mM at 25 ºC (Figure S1). Conversely, 2 disappears completely
in acetonitrile after 15 minutes at concentrations around 190 mM,
although it can be preserved in solution for longer times at lower
concentrations (Figure S2). Likewise, 2 is highly unstable in a
1:2 mixture of acetonitrile and water, even at low concentrations
(27 mM, Figure S3), which raises concerns about its potential
use for bioconjugation in physiological media.
Figure 1. The Ala-Ser-Gly triad in its open (a) and cyclic MIO form (b), in
histidine ammonia-lyase (HAL) from Pseudomonas putida (crystallographic
structures; PDB codes 1GK2 and 1EB4, respectively. Minimal (c) and
extended stapled peptides (d) used to attempt the chemical synthesis of MIO
scaffolds; the accepted mechanism for MIO formation involving intramolecular
cyclization and a double dehydration is shown for the minimal assayed peptide
(c).
Changing the lactam nitrogen atom of the MIO scaffold by an
oxygen –thus forming a lactone– is expected to preserve the
high reactivity of the native analogue. Along these lines, 4-
methylen-2-methyloxazol-5(4H)-one has been proposed to be a
key Michael acceptor intermediate in the biomimetic synthesis of
N-acetyl-4-bromotryptophan in route to clavicipitic acids.[13] In
further studies, such highly reactive type-2 alkene was found to
be formed in situ from serine and acetic anhydride and detected
by NMR spectroscopy in solution.[14] However, and unlike 4-
ethylidene- and specially 4-benzylidene-oxazol-5(4H)-ones,
which have been profusely synthetized and used in organic
synthesis,[15] the 4-methylidene analogues have remained very
elusive to both synthesis and application, due to their very low
stability.
The possibility to generate transient 4-methylen-oxazol-5(4H)-
ones as highly reactive Dha scaffolds for bioconjugation,
encouraged us to attempt their chemical synthesis. To this aim,
we first attempted the formation of the oxazol-5(4H)-one ring
using carbodiimides as coupling reagents from O-protected N-
acyl serine derivatives,[16] followed by base-promoted -
elimination. Thus, racemic N,O-dibenzoyl serine (1) was readily
obtained after treatment of DL-serine with excess BzCl under
basic aqueous conditions (unoptimized conditions, see
Supporting Information). After chromatographic purification,
5(4H)-oxazolone ring formation was assayed using N-(3-
Figure 1. (a) Synthesis of 4-methylen-2-phenyloxazol-5(4H)-one (MPO, 2)
from a conveniently protected serine (1). (b) 1H NMR spectra in CDCl3 (27 mM
for 1) showing the formation of compound 2 from starting material 1 at 3.7 and
15 min after the addition of DIC. (c) Consumption of starting material 1 (in
blue) and formation of compound 2 (in red) monitored by 1H NMR in CDCl3 (27
mM for 1). The initial time (t=0) represents the moment when DIC was added.
rt: room temperature.
dimethylaminopropyl)-N’-ethylcarbodiimide
(EDCI)
in
dichloromethane at 0 ºC, followed by an aqueous workup.
Although the starting material was completely consumed, no
identifiable product could be obtained. The 1H NMR spectrum of
the obtained material showed very broad signals likely
The influence of the protecting groups of serine on the reaction
outcome was then tested. With N-benzoyl-O-benzyl-DL-serine,
the fast formation of the oxazolone ring was also observed upon
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treatment with DIC. However, a notably slower β-elimination of
benzyl alcohol was observed, producing mixtures of the target
compound 2 and its cyclic precursor in variable ratios, since 2
decomposes over time. N-acetyl-O-benzyl-DL-serine was tested
with analogous results. Thus, N,O-dibenzoyl-DL-serine (1) was
selected as the more convenient starting material for the rest of
the study.
In situ generation of 2 from 1 in the presence of equimolecular
amounts of DIC, and subsequent addition of sulfur, nitrogen and
oxygen nucleophiles led to fast alkene decomposition. In all
biocompatible conditions,[23] as well as the manipulation of
steroelectronic effects of diazocompounds to increase their
reactivity and selectivity for bioorthogonal applications.[24]
Finally, uncatalyzed Diels-Alder cycloadditions with MPO 2 were
tested under the same conditions using various dienes such as
cyclohexadiene, cyclopentadiene, 2,3-dimethoxy-1,3-butadiene,
2,3-dimethyl-1,3-butadiene and 3,6-di-2-pyridyl-1,2,4,5-tetrazine
–the latter being commonly used for protein labelling via metal-
free strain-promoted inverse electronic demand Diels-Alder
reactions. Of note, cyclopentadiene was reactive enough to
cases, the desired conjugate addition reaction was not observed. cleanly react with freshly generated 2 to afford racemates endo-
With basic nucleophiles such as primary and secondary amines,
and thiolates and alkoxides generated in situ in the presence of
bases such as N,N-diisopropylethylamine or sodium hydride, 2
was completely degraded, likely through anionic polymerization
pathways. On the other hand, protonated thiols and alcohols
were not reactive enough to undergo conjugate addition during 2
lifetime at various concentrations.
6 and exo-6 in a 55:45 ratio. The structure of adduct exo-6 was
confirmed by X-ray diffraction analysis (Figure S8). Again, this
uncatalyzed and non-strain-promoted reaction with 2 proceeds
much faster at room temperature than with its acyclic analogue
MAA, which requires prolonged heating at around 100 ºC for 5 h
or the presence of a metal catalyst such as TiCl4.[25]
We then moved to test different reactions for which basic
conditions are not required (Scheme 1). Mukaiyama-Michael
conjugate addition with silyl enol ethers typically requires a
Lewis acid to take place.[17] However, after generating 2 in situ,
methyl trimethylsilyl dimethylketene acetal (MTDA) was added in
the absence of any catalyst. To our delight, the desired reaction
was completed in about 3 min leading to adduct 3 in moderate
yields after chromatographic purification. For comparison, the
same reaction with acyclic methyl 2-acetamidoacrylate (MAA)
takes 17 h in the presence of methyl aluminoxane as a Lewis
acid to achieve similar reaction yields.[18]
1,3-Dipolar cycloaddition reactions were then evaluated.
Addition of diazomethane to freshly generated 2 directly yielded
the spirocyclic cyclopropane
4 in 10 min. The common
pyrazoline intermediate whose ring-contraction via N2 extrusion
normally requires thermal or photochemical activation, such as
with
analogous
(Z/E)-4-benzyliden-2-phenyloxazol-5(4H)-
could be fully
ones,[19] was not observed. Compound
4
characterized by X-ray diffraction of monocrystals (Figure S6).
The reaction with ethyl diazoacetate (EDA) produced similar
results, leading to a mixture of racemic cyclopropanes 5a and 5b
in a nearly 1:1 ratio. Compound 5b could also be characterized
by X-ray diffraction analysis (Figure S7). MPO 2 clearly showed
a greater reactivity than related acyclic dehydroamino acid
analogues such as methyl 2-acetamidoacrylate towards
cyclopropanation with diazo compounds (Figures S4 and S5),
which normally require transition metal catalysts such as
rhodium or palladium to generate reactive metal carbene
species.[20] On the other hand, uncatalyzed 1,3-dipolar
cycloadditions with ethyl diazoacetate have been reported only
with highly activated substrates bearing nitro (α-carbethoxy-1-
nitrostyrenes and α-halo-α-nitroalkenes)[21] and nitrile groups
(arylidene–malononitrile and arylidene–ethyl cyanoacetate),[22]
although at very slow reaction rates (2-5 days needed).
Converserly, in situ generated compound 2 completely reacts
with diazo compounds in a few minutes. The second-order rate
constant for the reaction between 2 and EDA in CDCl3 at 25 ºC
was determined to be k2 = 3.9·10-3 M-1 s-1 by 1H NMR
spectroscopy (Figure S4), which is comparable to those found
for the 1,3-dipolar cycloadditions of strain-promoted alkynes.[23]
Recently, Raines and co-workers have described the selective
reaction of diazoacetamides with dehydroalanine residues in
Scheme 1. Fast reactions of MPO with different reagents under the same mild
conditions and without an extra activation.
The superior reactivity (i.e. electrophilicity) of cyclic MPO 2
compared to acyclic analogues such as methyl 2-
acetamidoacrylate can be rationalized by the large stabilization
of the LUMO of the former by 1.2 eV due to extensive
conjugation along the five-membered lactone and phenyl rings.
This translates into quite lower activation free energies (ΔG‡)
from 2 with respect to those from MAA, such as those calculated
quantum mechanically for the cycloaddition reactions with
diazomethane, methyl diazoacetate and cyclopentadiene
(Figures 3 and S9-S12). Regarding 1,3-dipolar cycloaddition
reactions with diazo compounds, a stepwise zwitterion-mediated
cyclopropanation mechanism with spontaneous nitrogen release
has been recently proposed[26] as an altenative pathway to the
commonly accepted asynchronous concerted cycloaddition
followed by nitrogen extrusion from the pyrazoline intermediates.
In fact, such stepwise mechanism was calculated to be
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significantly favored for compound 2, likely due to its ability to
delocalize the negative charge generated upon the Michael-type
addition of diazo compounds. Considering the large activation
energy required for the cleavage of the pyrazoline intermediates
(transition structures and intermediates could be calculated only
in the triplet excited state) compared to the retro-cycloaddition
reaction from the same intermediate, and given that the energy
barrier for the stepwise zwitterionic cyclopropanation reaction
between compound 2 and methyl diazoacetate is only ~2 kcal
mol-1 above the concerted transition state (Figure S10), such
process is likely to take place to some extent under the assayed
experimental conditions.
physiological conditions, although the low stability of MPO
derivatives in water currently limits their biological scope. This
possibility, together with the options to extend our methodology
to serine residues within a peptide context to chemically install
MIO-type modifications, are currently being evaluated in our
laboratory.
Experimental Section
General procedure for sequential one-pot synthesis of
2 and
subsequent reaction with different reagents. N,O-dibenzoylserine 1
(0.3 mmol) is introduced in a round bottomed flask and 10 mL of CHCl3 is
added. The heterogenous mixture is stirred at room temperature and
N,N’-diisopropylcarbodiimide (DIC, 0.3 mmol) is then added. The reaction
mixture dissolves immediately. Then, the reagent of interest (0.3 mmol) is
added and the mixture stirred for 3-30 min at room temperature. After
consumption of the starting material, the reaction mixture is transferred to
an extraction funnel and the organic layer is washed with 2 x 5 mL of
saturated NaHCO3 solution. Organic layers are combined, dried over
anhydrous Na2SO4 and the solvent evaporated. The reaction crude is
purified by VLC (hexane/AcOEt 100:0 to 80:20 gradient).
Acknowledgements
We thank funding from MINECO (projects CTQ2015-70524-R
and RYC-2013-14706 to G.J.O. and C.D.N.) and Universidad de
La Rioja (fellowship to N.M.).
Keywords: dehydroalanine • 4-methylen-oxazol-5(4H)-ones • 4-
methylideneimidazole-5-one • cycloadditions • diazo compounds
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Figure 3. Lowest energy transition structures calculated with
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with diazomethane (top), methyl diazoacetate (middle) and cyclopentadiene
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In summary, we have developed a simple methodology for the
synthesis of highly elusive 4-methylen-oxazol-5(4H)-ones with
aryl or alkyl groups at the 2-position, depending on the amine
protection of the starting serine derivative. Such cyclic
dehydroalanine derivatives are highly electrophilic and can be
quickly reacted in situ with silyl enol ethers, diazo compounds
and dienes under very mild conditions at room temperature, and
in the absence of metal catalysts without the need of ring strain
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Carlos Aydillo, Nuria Mazo, Claudio D.
Navo, and Gonzalo Jiménez-Osés*
Page No. – Page No.
Elusive Dehydroalanine Derivatives
with Enhanced Reactivity
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COMMUNICATION
Carlos Aydillo, Nuria Mazo, Claudio D.
Navo, and Gonzalo Jiménez-Osés*
Page No. – Page No.
Elusive Dehydroalanine Derivatives
with Enhanced Reactivity
For the first time, a highly reactive and elusive dehydroalanine scaffold has been
prepared, and quickly reacted in situ with a variety of reagents without any type of
extra activation, paving the way for bioconjugation applications.
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