Design and synthesis of novel 19F-amino acid: A promising 19F NMR label for peptide studies

Article abstract of DOI:10.1021/ol503300m

Novel aliphatic ¹⁹F-substituted amino acid was designed as a ¹⁹F NMR label for peptide studies. The synthesis was performed in 11 steps and 9% overall yield from a commercially available starting material. The key transformation was a decarboxylative fluorination of an aliphatic carboxylic acid with XeF₂ in C₆F₆.

Full text of DOI:10.1021/ol503300m

Letter
pubs.acs.org/OrgLett
1
9
19
Design and Synthesis of Novel F‑Amino Acid: A Promising F NMR
Label for Peptide Studies
Dmytro Bandak,†,⊥ Oleg Babii, Roman Vasiuta, Igor V. Komarov, and Pavel K. Mykhailiuk*
‡
†,#
§
,†,∥
†
Department of Chemistry, Taras Shevchenko National University of Kyiv, Volodymyrska 64, Kyiv 01601, Ukraine
‡
Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, Karlsruhe 76131, Germany
§
Institute of High Technologies, Taras Shevchenko National University of Kyiv, Volodymyrska 60, Kyiv 01601, Ukraine
∥Enamine, Ltd., Oleksandra Matrosova Street 23, Kyiv 01103, Ukraine
*
S Supporting Information
ABSTRACT: Novel aliphatic 19F-substituted amino acid was designed
19
as a F NMR label for peptide studies. The synthesis was performed in
1 steps and 9% overall yield from a commercially available starting
1
material. The key transformation was a decarboxylative fluorination of
an aliphatic carboxylic acid with XeF in C F .
2
6 6
olid-state 19F NMR spectroscopy is a useful technique to
with its aliphatic isostere. In drug discovery, chemists commonly
use three saturated bioisosters of benzene: bicyclo[1.1.1]-
S
study membrane-active peptides under their natural
1
5,6
7
8
conditions in lipid bilayers. In this method, scientists first
selectively incorporate a suitable fluorine-containing amino acid
pentane,
bicyclo[2.2.2]octane, and cubane. All three
compounds have collinear alignment of the substituents and
19
(
F label) into the peptide of interest. Several very strict criteria
similar C−C distances (Figure 1). In this work, we report our
19
9
to an effective F label exist, however. The amino acid must (a)
be conformationally constrained to place the 1 F reporter in a
well-defined position to aminocarboxylic moiety; (b) be
compatible with the peptide synthesis; and (c) not change
studies on the bicyclo[2.2.2]octane skeleton. We envisioned
9
that such replacement should make the resulting aliphatic amino
acid 2 less prone to racemization than 1 due to the absence of the
stabilizing effect of the benzene ring on the enolate ion.
1
structure and function of the peptide.
19
2
3
F labels bearing either CF or CF groups have been used in
3
2
a number of peptide studies. Mono-F-substituted amino acids,
however, have received less attention. Recent validation of (4-
1
9
4
fluorophenyl)glycine (1) (Figure 1) as a F label, for example,
revealed several drawbacks. First, the amino acid 1 completely
racemized during the peptide synthesis, making thereby an
assignment of the stereoconfiguration of the obtained epimeric
peptides (R or S) extremely problematic. In addition, replacing
the residues of aliphatic amino acids (Ala, Val, Ile, Leu) with
aromatic compound 1 changed the structure/function of some
peptides. It is not surprising, therefore, that amino acid 1 has not
received wide popularity in peptide studies as a F label.
In this work, we have rationally designed and synthesized an
aliphatic analogue of 1 which is less prone to racemizationthe
amino acid 2 (F-Bog).
Figure 1. Isosteric cores: benzene and bicyclo[2.2.2]octane.
Retrosynthetic Analysis
1
9
We planned to synthesize the chiral amino acid 2 from the
corresponding aldehyde 3 using the Strecker reaction with a
chiral auxiliary (chiral amine) followed by a removal of the
protecting groups (Scheme 1). We envisioned that aldehyde 3, in
turn, could be prepared from the known ester 4.
Scheme 1. Retrosynthetic Analysis of Amino Acid 2
Design
Amino acid 1 racemized during the peptide synthesis because of
the structural features contributing to the stability of the
corresponding enolate ion, an adjacent benzene ring, and
efficient electron-withdrawing effect of the fluorine atom easily
transmitted via the benzene ring to the aminocarboxylate moiety.
Therefore, we came up with an idea of replacing the benzene ring
Received: November 13, 2014
©
XXXX American Chemical Society
A
DOI: 10.1021/ol503300m
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Synthesis
Scheme 3. Synthesis of Amino Acid 4
In 1982, Adcock et al. synthesized 4 from ethyl acetoacetate via
10
acid 5 in 10 steps (Scheme 2). The final fluorination of alcohol 6
1
0,12
Scheme 2. Literature Syntheses of Compound 4.
was performed with SF . The use of highly toxic gas SF , however,
4
4
limits the applicability of this method. In addition, storage and
handling of SF is forbidden in some European countries and in
4
11
the United States. Later, Della et al. performed an alternative
synthesis of compound 4 from ethyl acetoacetate, but still in nine
1
2
steps. The key transformation was a reaction of iodide 7 with
XeF . We decided, therefore, to develop a shorter synthetic
2
strategy to 4 from the same starting materials.
We started the synthesis of 4 from the commercially available
diester 8 as a mixture of isomers (Scheme 3). Double alkylation of
a
Key: (a) LDA (1.2 equiv), HMPA (4 equiv), THF, −78 °C; (b)
BrCH CH Cl (1.1 equiv), −78 °C; (c) LDA (1.2 equiv), THF, −78
2
2
8
with BrCH CH Cl using LDA/HMPA afforded the bicyclic
°C; (d) NaOH (1.0 equiv), MeOH/H O (2/1), 20 °C; (e) XeF (1.5
2
2
2
2
8a,13
derivative 9 in 72% yield following the literature procedure.
Monohydrolysis of the ester group in 9 using 1 equiv of NaOH in
methanol/water mixture gave acid 10 in 55% yield. Finally, the
crucial step, conversion of the carboxylic group in 10 into the
fluorine atom with XeF2, was attempted. The reaction in
CH Cl afforded a mixture of the target fluoride 4 and the side
product 11, however. The source of the hydrogen atom in
formation of 11 was presumably the solvent. To prevent
incorporation of the hydrogen atom into the product, we decided
to attempt the reaction in a polyfluorinated solvent. In fact, the
reaction of 10 with XeF in C F gave 4 as the sole product in 68%
equiv), CH
2
Cl
2
, 0 °C; (f) XeF
2
(1.5 equiv), C
3 3
6
F
6
, reflux; (g) LiAlH
4
(2.5 equiv), THF, reflux; (h) SO ·Py (3.6 equiv), NEt (3.0 equiv),
14
CH Cl , DMSO, −10 → 20 °C; (i) (R)-2-phenylglycinol (1.0 equiv),
2
2
Me SiCN (3.0 equiv), CH Cl /MeOH (1/1), 0 °C; (j) MeOH, reflux;
3
2
2
15
(k) column chromatography; (l) Pb (OAc) (1.5 equiv), CH Cl /
2 4 2 2
MeOH (2/1), 0 °C; (m) 2 N HCl, reflux; (n) Ac O (1.5 equiv),
2
2
2
CH Cl , 20 °C; (o) 6 N HCl, reflux; (p) cation-exchange
6
2
2
chromatography on KU-2 resin.
solution of 13/14 in MeOH at reflux for 3 h, the isomerization
reached the thermodynamic equilibrium of 13/14 = 90/10. This
ratio did not change after the subsequent heating. At this point,
we isolated the needed major isomer 13 from the reaction
mixture by column chromatography on silica gel in 84% overall
yield (from 12 in four steps). The needed (S)-stereoconfigura-
tion of the newly formed chiral center in 13 was confirmed by X-
ray crystallographic analysis (Figure 2). Oxidative cleavage of the
chiral auxiliary in 13 with Pb(OAc) in CH Cl followed by the
2
6 6
yield after sublimation. Formation of the side compound 11 was
not observed. The developed transformation was scalable, and we
synthesized ca. 7 g of pure ester 4.
Next, we performed reduction of ester 4 with LiAlH into
4
alcohol 12. Oxidation of the hydroxylic group in 12 using Py·SO3
complex in DMSO afforded the volatile aldehyde 3 that was used
in the next step without isolation. To perform the planned
Strecker synthesis with a chiral auxiliary, we focused our attention
at the known one, α-phenylglycinol. Because of the presence of
polar hydroxy group, the diastereomeric Strecker products can
often be separated by column chromatography on silica gel more
efficiently compared with the corresponding derivatives of α-
phenylethylamine. Unfortunately, Strecker reaction of aldehyde
4
2
2
acidic hydrolysis of the intermediate Schiff base gave nitrile 15.
Unexpectedly, we were not able to hydrolyze the nitrile group in
15 under acidic conditions: the reaction did not proceed even in
40% aq HBr under reflux (only the intact starting material was
isolated). Apparently, the bulky bicyclo[2.2.2]-octyl substituent
in 15 hampered the reaction. To overcome this problem, we
decided to attempt an acidic hydrolysis of N-acetylated derivative
16 with the intent that the carbonyl group would facilitate the
reaction via intramolecular anchimeric interaction with the nitrile
16
3
1
with (R)-α-phenylglycinol gave a mixture of stereoisomers 13/
4 in an almost equimolar ratio of 57/43. We noticed, however,
that at room temperature in MeOH this ratio slowly changed in a
favor of 13. Obviously, 14 underwent isomerization into 13.
Presumably, this process occurred via the MeOH-separated
contact ion pair of 14. Absence of isomerization in less polar
hexane supported this suggestion. We found that the isomer-
ization proceeded faster at higher temperature: after heating a
(Scheme 3, blue arrow). In the event, acylation of 15 with Ac O
2
smoothly proceeded in CH Cl without base at room temper-
2
2
17
ature to give 16. As expected, hydrolysis of 16 proceeded easily in
6 N HCl during 2 h to give the target amino acid 2 in 77% yield
after ion-exchange chromatography.
B
DOI: 10.1021/ol503300m
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
the residue of (S)-2, while the minor one, 18b, contained the
residue of (R)-2.
In summary, we have synthesized novel conformationally rigid
fluorinated α-amino acid 2. This compound was rationally
designed as an aliphatic analogue of the prone-to-racemization
19
F label 1. In fact, amino acid 2 was compatible with the standard
protocol of SPPS and racemized only partially, therefore solving
the problem of determination of stereoconfiguration of the
obtained epimeric peptides. This makes amino acid 2 an
attractive 19F label to replace the residues of aliphatic amino
1
9
acids (Val, Ile, Leu) in peptides for their study via F NMR.
Incorporation of 2 into naturally occurring peptides for
subsequent NMR studies is ongoing.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Experimental procedures and copies of NMR spectra. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Figure 2. Molecular structure of compound 13 according to X-ray
diffraction data (F, red; N, blue; O, orange).
18
Finally, we wanted to check whether amino acid 2 would
indeed be more stable to racemization than 1. Having observed
that acidic hydrolysis of 15 was not effective, we feared that
sterically restricted amino acid 2 would not be compatible with
the standard protocol of solid phase peptide synthesis (SPPS).
Therefore, we first synthesized an Fmoc-protected derivative 17
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: pavel.mykhailiuk@gmail.com, pavel.mykhailiuk@mail.
enamine.net.
Present Addresses
(
Scheme 4), and attempted next the synthesis of simple
⊥(
D.B.) Department of Chemistry and Biochemistry, University
of Arizona, 1306 E. University Blvd, Tucson, AZ.
R.V.) Department of Organometallic Chemistry, Petersen-
a
Scheme 4
#
(
strasse 18, Technische Universitaet Darmstadt, 64287 Darm-
stadt, Germany.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Prof. O. Shishkin (Institute for Single Crystals,
Kharkiv, Ukraine) for X-ray analysis of nitrile 13, to Dr. O.
Petrenko (Enamine Ltd., Kyiv, Ukraine) for the starting batch of
ester 9, to Prof. A. Tolmachev (Enamine Ltd., Kyiv, Ukraine) for
financial support, to Dr. S. Afonin (KIT, Karlsruhe, Germany) for
MALDI-measurements, to Dr. M. Gorichko (Taras Shevchenko
National University of Kyiv, Kyiv, Ukraine) for the idea to use
C F in the synthesis of 4, and to Dr. O. Buravov (Enamine Ltd.,
a
6 6
Reagents and conditions: (a) FmocCl (1.1 equiv), K CO3 (2.0
2
Kyiv, Ukraine) for the advice on the hydrolysis of nitrile 15 via
previous N-acylation.
equiv), dioxane/water, 20 °C); solid-phase peptide synthesis (starting
from alanine preloaded 2-chlorotrityl resin), 17 (1.2 equiv), HOBt
(
2
1.2 equiv), PyBOP (1.2 equiv), DIPEA (2.4 equiv); (c) 20 °C; TFA,
0 °C; (d) HPLC separation.
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DOI: 10.1021/ol503300m
Org. Lett. XXXX, XXX, XXX−XXX