Pedro P de Castro1, Isabela M R Rimulo1, Angelina M de Almeida1, Renata Diniz2, Giovanni W Amarante1. 1. Department of Chemistry, Federal University of Juiz de Fora, Campus Martelos, Juiz de Fora, Minas Gerais 36036-900, Brazil. 2. Department of Chemistry, Federal University of Minas Gerais, Campus Pampulha, Belo Horizonte, Minas Gerais 31270-901, Brazil.
Abstract
An organocatalytic protocol, employing the commercially available EDC as coupling agent, has been developed for the preparation of dual-protected amino acid derivatives without epimerization. This methodology was then applied to different Boc-amino acid and amine derivatives in moderate to excellent isolated yields. In addition, racemization-free Boc deprotection was also demonstrated. Mechanism investigation through electrospray ionization (+)-mass spectrometry/mass spectrometry revealed an acyclic intermediate (no azlactone formation) activated by the camphorsulfonic acid as an organocatalyst as a key step for the sequential attack of the nucleophile.
An organocatalytic protocol, employing the commercially available EDC as coupling agent, has been developed for the preparation of dual-protected amino acid derivatives without epimerization. This methodology was then applied to different Boc-amino acid and amine derivatives in moderate to excellent isolated yields. In addition, racemization-free Boc deprotection was also demonstrated. Mechanism investigation through electrospray ionization (+)-mass spectrometry/mass spectrometry revealed an acyclic intermediate (no azlactone formation) activated by the camphorsulfonic acid as an organocatalyst as a key step for the sequential attack of the nucleophile.
Epimerization of stereocenters
are a common and generally undesirable
process in organic synthesis.[1−3] This phenomenon is especially
relevant in the stereoselective preparation of amino acid derivatives,
due to the presence of acidic α-carbonyl hydrogens in their
scaffold.[4−6] Several complex coupling agents for the racemization-free
formation of amino acid amides and peptides have recently been developed[7−10] (Scheme A–C).
Scheme 1
Previous Reports on Epimerization-Free Preparation of Amino Acid
Amides
This problem is particularly
important in transformations involving
azlactones (also known as oxazolones).[11−16] These heterocycles are basically protected and cyclic amino acids
bearing a very acidic hydrogen (pKa ≈
9) in their scaffolds,[17] which leads to
fast racemization. Our research group has been extensively studying
the reactivity of azlactones in various transformations[18−24] including racemic amide formation through the azlactone ring-opening
reaction,[25] and recently we have been interested
in the development of an epimerization-free methodology to synthesize
these cycles.To the best of our knowledge, only one research
group has described
the racemization-free preparation of oxazolones.[26,27] It was shown that the presence of EDC, N-Boc, or N-Cbz-amino acids affords enantiomerically pure 2-alcoxy-azlactones.
A few years later, Bagno’s group reported the first crystal
structures of 2-alcoxy-oxazolones from Fmoc and Cbz, however, without
α-carbonyl hydrogens.[28] On the other
hand, recent studies have described the racemization of azlactones
from carbamates.[29,30]In this context, we herein
present an efficient and epimerization-free
method for the preparation of dual-protected amino acid derivatives
employing the commercially available EDC as a coupling agent (Scheme D). We also provide
new insights to explain the racemization-free nature of this transformation,
showing that despite having the necessary scaffold for the cyclization, N-Boc protected amino acids do not form an azlactone when
activated by EDC.
Results and Discussion
In our attempt
to prepare 2-alcoxy-oxazolone derivatives, N-Boc-amino
acids were chosen as substrates. Initially, l-isoleucine,
which contains 2 stereocenters, and therefore
the epimerization of the α-carbonyl hydrogen would result in
a pair of diastereomers, was chosen as an ideal substrate. This, in
fact, would allow us to observe racemization from the crude reaction
mixture 1H NMR analyses.During the reaction of N-Boc-l-isoleucine
with EDC, the formation of an unstable intermediate was observed by
thin-layer chromatography (TLC). Due to this instability, the crude
reaction mixture was washed with distilled cold water three times,
and the dichloromethane immediately employed, without further purification,
in a sequential reaction in the presence of pyrrolidine as nucleophile.
To our delight, the desired product 2ae was isolated
in 38% yield and without apparent epimerization. After new attempts
to increase the general yields, it was found that the use of 10 mol
% of (±)-camphorsulfonic acid (CSA) as organocatalyst resulted
in an increase to 73% yield.Having established this catalytic
system for the epimerization-free
formation of the amino acid amide derivative, we proceeded to test
the generality of these reaction conditions with a range of different
Boc-amino acids and amines. Initially, primary aliphatic and aromatic
amines were examined, providing products 2a–p in
yields ranging from 52 to 81% (Scheme ). Anilines also worked well, providing 2m and 2n in 73–76% yields. Even the use of ortho-substitutedaniline was well tolerated, affording 2o in 70% yield.
The heteroaromatic 2-amino-3-picoline proved to be reactive under
the developed reaction conditions, providing 2p in a
52% isolated yield.
Scheme 2
Epimerization-Free Preparation of Amino Acid Amides
Employing Primary
Amines as Nucleophiles
Next, we turned our attention toward the secondary amines
as nucleophiles.
Compounds 2q–ag were isolated in yields ranging
from 61 to 86% (Scheme ), with no need of purification by the chromatographic column. Different
Boc-amino acids could also be employed, with no significant influence
on the reaction yield.
Scheme 3
Epimerization-Free Preparation of Amino
Acid Amides Employing Secondary
Amines as Nucleophiles,
Compounds 2s and 2y are, respectively, the racemic mixtures of 2r and 2x.
Reaction
also carried out on a gram scale.
Epimerization-Free Preparation of Amino
Acid Amides Employing Secondary
Amines as Nucleophiles,
Compounds 2s and 2y are, respectively, the racemic mixtures of 2r and 2x.Reaction
also carried out on a gram scale.It is worth
mentioning that no epimerization was observed by NMR
for any of l-isoleucine derivatives. The relative and absolute
stereochemistry of the compound 2m was determined by
the X-ray crystallographic structure (Figure A), with maintenance of the original (2S,3S)-isoleucine stereocenters. Compound 2x and its racemic mixture 2y, prepared using d,l-alanine were also evaluated by chiral high-performance
liquid chromatography (HPLC) (Figure B), providing another insight into the epimerization-free
nature of this transformation.
Figure 1
A) X-ray crystallographic structure of 2m (anisotropic
displacement ellipsoids are drawn at the 50% probability level) (B)
Chiral HPLC of 2x and its racemic mixture, 2y.
A) X-ray crystallographic structure of 2m (anisotropic
displacement ellipsoids are drawn at the 50% probability level) (B)
Chiral HPLC of 2x and its racemic mixture, 2y.We also decided to investigate
the racemization process during
Boc deprotection. Employing classical reaction conditions,[31] products 2ac and 2q in the presence of TFA afforded the desired salts 2ag and 2ah in 86 and 84% yield (Scheme ).
Scheme 4
Epimerization-Free Boc Deprotection
of Compounds 2q and 2ac
We then turned our attention toward the understanding
of the basics
of this reaction by investigating the plausible key intermediate.
To this end, two experiments were carried out in triplicate employing
Boc-l-isoleucine and EDC. First, a crude reaction mixture
was transferred to the gas phase by electrospray ionization (ESI),
followed by (tandem) mass spectrometry (MS) characterization ESI(+)-MS/MS.
The other experiment consisted of analyzing the same sample after
the dichloromethane was washed with distilled water (for the removal
of EDC and its urea) and dried under reduced pressure. Surprisingly,
both samples showed no sign of an azlactone intermediate; however,
an ion of m/z 387.2971 was found
in both experiments and was extremely abundant in the purified sample
(see Supporting Information).This
key species of m/z 387 corresponds
to intermediate 1 (Figure ), the Boc-l-isoleucine activated by EDC.
We propose that due to a torsional problem, different from Fmoc or
Cbz-amino acids with previously reported X-ray crystallographic structures,[28] the tert-butoxy group prevents
the intramolecular cyclization and azlactone formation. Moreover,
because the pKa of an α-carbonyl
hydrogen of an azlactone[32,33] is considerably lower
than that of an acyclic amino acid,[34] this
helps to explain the epimerization-free nature of this reaction.
Figure 2
ESI(+)-MS
of crude reaction between Boc-l-isoleucine and
EDC.
ESI(+)-MS
of crude reaction between Boc-l-isoleucine and
EDC.Finally, the organocatalytic mechanism
was investigated by ESI(+)-MS/MS.
For this purpose, the previous crude reaction sample bearing ion m/z 387 was added to 10 mol % of the CSA
catalyst and immediately injected into the mass spectrometer. Two
ions of m/z 641.3555 and of m/z 657.3298 corresponding to the association
of the intermediate 1 and the catalyst, that is, [M + Na]+ and [M + K]+ were intercepted (Figure A). The collision-induced dissociation of m/z 641 shows fragment ions of m/z 387 and 409 (Figure B), corresponding to the intermediate 1 [M + H]+ and [M + Na]+, due to the
neutral loss of CSA. Therefore, as previously reported for other Brønsted
acid catalysts,[35] the mechanism seems to
proceed through an ion-pairing intermediate that is then followed
by amine nucleophilic attack.
Figure 3
(A) ESI(+)-MS of intermediate 1 in the presence of
CSA as catalyst (expansion of the ion of m/z 641); (B) ESI(+)-MS/MS (15 eV) of the ion of m/z 641.
(A) ESI(+)-MS of intermediate 1 in the presence of
CSA as catalyst (expansion of the ion of m/z 641); (B) ESI(+)-MS/MS (15 eV) of the ion of m/z 641.
Conclusions
In summary, general conditions have been reported
for the organocatalytic
and epimerization-free preparation of dual-protected amino acid derivatives
under mild reaction conditions. A broad substrate scope has been presented,
allowing changes in the amino acid and the amine nucleophile, proving
the generality of this protocol. Boc deprotection was also carried
out in excellent yields and without racemization. ESI(+)-MS/MS experiments
revealed an acyclic intermediate, unlike azlactone formation. Finally,
the organocatalytic mechanism investigation revealed intermediate
activation by the catalyst CSA to form an ion-pairing intermediate,
that is then followed by nucleophilic attack.
Experimental Section
General
Methods
All purchased chemicals were used as
received without further purification. Solvents were dried according
to standard procedures. Analytical TLC was performed on TLC plates
(silica gel 60 F254) and visualized employing a ninhydrin (2,2-dihydroxyindane-1,3-dione)
indicator. The chemical shifts are reported in ppm relative to the
solvent residual peak. The 1H NMR spectra were recorded
at 500 MHz, 13C NMR spectra were recorded at 125 MHz. Chemical
shifts are reported in ppm using the following peak pattern abbreviations:
br, broad; s, singlet; d, doublet; t, triplet; q, quartet; pent, pentet;
sext, sextet; m, multiplet. High-resolution mass spectra, as well
as mechanism investigation were investigated in the positive ion mode
using a time-of-flight (TOF) mass spectrometer equipped with an ESI
source or a matrix-assisted laser desorption (MALDI)/TOF spectrometer
using a pulsed nitrogen laser at 337 nm. Single-crystal data for compound 2m were collected with a diffractometer with Mo Kα (λ
= 0.71073 Å) radiation at room temperature (298 K). The data
collection, cell refinements, and data reduction were performed using
the CRYSALISPRO software.[35] To elucidate the structure, an empirical absorption
correction using the faces’ crystal model was employed. The
structures were resolved by direct methods and refined by full-matrix
least square refinement on F2 using the SHELXL-2014 program package, and all nonhydrogen atoms were refined
with anisotropic thermal parameters. H atoms connected to carbon were
placed in idealized positions and treated by the rigid model, with
Uiso(H) = 1.2 Ueq(C) for the aromatic ring and Uiso(H) = 1.5 Ueq(C)
for methyl groups. CCDC 1539443 has the supplementary crystallographic
data for this work, and it can be obtained free of charge from the
Cambridge Crystallographic Data Centre at http://www.ccdc.cam.ac.uk/conts/retrieving.html. The figures were drawn using ORTEP-3 for Windows and Mercury programs.
Enantiodiscriminating HPLC was performed using a 4.6 × 25 cm2 Chiralpak IA column (isocratic elution using hexane/isopropanol
88:12; flow 0.5 mL/min). Melting points and optical rotations were
recorded, respectively, on a melting point apparatus and a polarimeter.
General Method: Organocatalyzed Preparation of Amino Acids Amides
In a round-bottom flask, N-Boc-amino acid (i.e., l-alanine, l-phenylalanine, l-valine, l-leucine, or l-isoleucine) (1.0 mmol) and EDC·HCl
(211 mg, 1.1 mmol) were dissolved in dichloromethane (15.0 mL), and
the reaction was stirred at 0 °C for 30 min. The crude reaction
was washed with distilled water three times, and the dichloromethane
immediately employed, without further purification. This solution
was then transferred to a round-bottom flask containing the amine
nucleophile (2.0 mmol) and the (±)-camphorsulfonic acid (23 mg,
10 mol %). After 24 h, the reaction was concentrated in vacuo, and
for l-isoleucine derivatives, an aliquot was taken to the
NMR spectrometer for analysis of the epimerization by 1H NMR analysis. The desired products were then purified by recrystallization
in hot EtOH/water, liquid–liquid extraction, or column chromatography.
General Method: Boc Deprotection of Amino Acids Amides
In
a round-bottom flask, Boc-l-amino acid amide (1.0 mmol,
1.0 equiv) was dissolved in anhydrous CH2Cl2 (10 mL), and TFA (1 mL, 22.0 equiv) was added. After 3 h stirring
at room temperature, the reaction was concentrated in vacuo, directly
affording the unprotected product as a yellow/orange oil.
Authors: Albrecht Berkessel; Felix Cleemann; Santanu Mukherjee; Thomas N Müller; Johann Lex Journal: Angew Chem Int Ed Engl Date: 2005-01-21 Impact factor: 15.336
Authors: Eloah P Ávila; Rodrigo M S Justo; Vanessa P Gonçalves; Adriane A Pereira; Renata Diniz; Giovanni W Amarante Journal: J Org Chem Date: 2014-12-12 Impact factor: 4.354
Authors: Zhao Wu; Joshua D Laffoon; Trang T Nguyen; Jacob D McAlpin; Kami L Hull Journal: Angew Chem Int Ed Engl Date: 2016-12-27 Impact factor: 15.336
Scheme 1
Scheme 2
Scheme 3
Figure 1
Scheme 4
Figure 2
Figure 3