Chemoenzymatic synthesis of (S)-hexafluoroleucine and (S)- tetrafluoroleucine

Article abstract of DOI:10.1021/ol702470j

We have developed a short chemoenzymatic synthesis for both (S)-5,5,5,5′,5′,5′-hexafluoroleucine (Hfl) and (S)-5,5,5′,5′-tetrafluoroleucine (Qfl) on gram scale. Qfl was incorporated into a peptide using standard solid-phase peptide synthesis protocols to measure its helix propensity. The helix propensity for Qfl is 0.68 kcal·mol^-1 more favorable compared to Hfl.

Full text of DOI:10.1021/ol702470j

ORGANIC
LETTERS
2
007
Vol. 9, No. 26
517-5520
Chemoenzymatic Synthesis of
5
(S)-Hexafluoroleucine and
S)-Tetrafluoroleucine
(
Hsien-Po Chiu and Richard P. Cheng*
Department of Chemistry, UniVersity at Buffalo, The State UniVersity of New York,
Buffalo, New York 14260-3000
chengr@buffalo.edu
Received October 10, 2007
ABSTRACT
We have developed a short chemoenzymatic synthesis for both (S)-5,5,5,5′,5′,5′-hexafluoroleucine (Hfl) and (S)-5,5,5′,5′-tetrafluoroleucine (Qfl)
on gram scale. Qfl was incorporated into a peptide using standard solid-phase peptide synthesis protocols to measure its helix propensity.
-1
The helix propensity for Qfl is 0.68 kcal‚mol more favorable compared to Hfl.
Highly fluorinated amino acids have been used to stabilize
diastereomeric mixtures of (2S)-5,5,5-trifluoroleucine (Tfl)1,2,5
proteins1
-10
for potential applications in industrial scale
or enantiomerically pure (S)-5,5,5,5′,5′,5′-hexafluoroleucine
11
3,6,8,9
biotransformations, biosensors, and protein therapeutics. In
particular, leucine (Leu) has been replaced with either
(Hfl)
to stabilize helical proteins (Figure 1). Despite the
(
1) Tang, Y.; Ghirlanda, G.; Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard,
W. A., III; DeGrado, W. F.; Tirrell, D. A. Biochemistry 2001, 40, 2790-
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2
(
F.; Tirrell, D. A. Angew. Chem., Int. Ed. 2001, 40, 1494-1496.
(
(
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1
257.
(
5) Bilgi c¸ er, B.; Fichera, A.; Kumar, K. J. Am. Chem. Soc. 2001, 123,
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393-4399.
(
(
Figure 1. Leucine and fluorinated leucine analogues.
6) Bilgi c¸ er, B.; Kumar, K. Tetrahedron 2002, 58, 4105-4112.
7) Horng, J.-C.; Raleigh, D. P. J. Am. Chem. Soc. 2003, 125, 9286-
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(
8) Lee, K.-H.; Lee, H.-Y.; Slutsky, M. M.; Anderson, J. T.; Marsh, E.
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Chem. Soc. 2006, 128, 337-343.
10) Woll, M. G.; Hadley, E. B.; Mecozzi, S.; Gellman, S. H. J. Am.
various published procedures for synthesizing Hfl,3
,12-14
the
(
generation of enantiomerically pure Hfl remains challenging.
Furthermore, we recently showed that the helix propensity
decreases drastically (>1 kcal‚mol ) upon replacing Leu
(
-
1
Chem. Soc. 2006, 128, 15932-15933.
(
11) Ritter, S. K. Chem. Eng. News 2007, 85(37), 36-37.
(12) Zhang, C.; Ludin, C.; Eberle, M. K.; Stoeckli-Evans, H.; Keese, R.
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(
13) Xing, X.; Fichera, A.; Kumar, K. Org. Lett. 2001, 3, 1285-1286.
1
0.1021/ol702470j CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/29/2007

1
5
propensity.18-23 Three, the amino acid needs to be readily
synthesized in enantiomerically pure form. Four, the amino
acid needs to be readily incorporated into peptides through
solid-phase peptide synthesis. Toward fulfilling these needs,
herein we report the gram scale stereoselective synthesis of
both Hfl and a novel fluorinated amino acid (S)-5,5,5′,5′-
tetrafluoroleucine (Qfl, Figure 1), the incorporation of Qfl
into peptides, and the helix propensity of Qfl.
with Hfl, suggesting the need to develop novel fluoro-amino
acids with more favorable helix propensities compared to
Hfl.
Scheme 1. Chemoenzymatic Synthesis of Hfl and Qfl.
The difluoromethyl group (CF
the development of bioactive compounds.
has been used as a bioisostere for the hydroxyl group because
2
H) has been exploited in
2
4-31
This group
32
of similar hydrogen bond donating capability. Nonetheless,
the CF H group was incorporated as difluoromethionine into
the interior of a protein to probe the environment near the
2
3
3
fluorines, demonstrating the compatibility of CF
2
H with
the hydrophobic core of proteins. If two CF H groups were
2
introduced to give Qfl, the overall shape of Qfl would be
similar to Leu without introducing any additional stereo-
center. Furthermore, the size of Qfl would be in between
that of Leu and Hfl, both of which are compatible with helical
3
,6,8,9
proteins.
We synthesized Hfl and Qfl on gram scale using a
15
chemoenzymatic approach (Scheme 1). The corresponding
acetones were reacted with the ylide in a Wittig reaction to
give the unsaturated pyruvate esters (1a and 1b). For
synthesizing 1a, we consistently obtained the side product
An ideal fluoro-amino acid substitute for Leu should have
the following characteristics. One, the overall shape of the
amino acid needs to be similar to Leu, because Koksch has
shown that the shape is important for substituting Leu
residues in coiled coils.16,17 Two, the helix propensity needs
to be similar to Leu, or at least needs to be more favorable
than Hfl. Unfortunately, the design of fluoro-amino acids
5
(g30%) (Figure 2), despite performing the reaction
1
5
with reasonable helix propensity remains difficult despite
the extensive knowledge of the main determinants for helix
(
15) Chiu, H.-P.; Suzuki, Y.; Gullickson, D.; Ahmad, R.; Kokona, B.;
Fairman, R.; Cheng, R. P. J. Am. Chem. Soc. 2006, 128, 15556-15557.
16) J a¨ ckel, C.; Salwiczek, M.; Koksch, B. Angew. Chem., Int. Ed 2006,
5, 4198-4203.
17) J a¨ ckel, C.; Seufert, W.; Thust, S.; Koksch, B. ChemBioChem 2004,
, 717-720.
18) Creamer, T. P.; Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
(
Figure 2. Chemical structures of two side products in the
chemoenzymatic synthesis of Hfl.
4
5
5
(
(
937-5941.
according to literature procedures (1 equiv of hexfluoroac-
(
19) Yang, A.-S.; Honig, B. J. Mol. Biol. 1995, 252, 351-365.
(20) Luo, P.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
etone, 4.0 M ylide in tetrahydrofuran, 55 °C, vacuum
4
930-4935.
21) Garc ´ı a, A. E.; Sanbonmatsu, K. Y. Proc. Natl. Acad. Sci. U.S.A.
002, 99, 2782-2787.
22) Vila, J. A.; Ripoll, D. R.; Scheraga, H. A. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 13075-13079.
23) Avbelj, F.; Luo, P.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A.
12
distillation). Hexafluoroacetone gas and hexafluoroacetone
(
2
hexahydrate gave virtually the same results. To minimize
this side product, we changed the equivalents of hexafluo-
roacetone, concentration of the ylide, solvent, and reaction
temperature. By using 1.5 equiv of hexafluoroacetone under
dilute conditions (0.4 M ylide) in benzene at 40 °C, the
desired product was obtained with >99% purity upon
vacuum distillation with 65% isolated yield. Either more
equivalents of hexafluoroacetone or lower reaction temper-
atures resulted in a slight drop in isolated yield, whereas
(
(
2
1
1
000, 97, 10786-10791.
(
24) Wang, E. A.; Walsh, C. Biochemistry 1981, 20, 7539-7546.
(25) Bey, P.; Gerhart, F.; van Dorsselaer, V.; Danzin, C. J. Med. Chem.
983, 26, 1551-1556.
(
(
26) Imperiali, B.; Abeles, R. H. Biochemistry 1986, 25, 3760-3767.
27) Yamazaki, T.; Haga, J.; Kitazume, T. Bioorg. Med. Chem. Lett.
991, 1, 271-276.
28) Pu, Y. M.; Torok, D. S.; Ziffer, H.; Pan, X.-Q.; Meshnick, S. R. J.
Med. Chem. 1995, 38, 4120-4124.
29) Narjes, F.; Koehler, K. F.; Koch, U.; Gerlach, B.; Colarusso, S.;
(
(
Steink u¨ hler, C.; Brunetti, M.; Altamura, S.; De Francesco, R.; Matassa, V.
(33) Vaughan, M. D.; Cleve, P.; Robinson, V.; Duewel, H. S.; Honek,
J. F. J. Am. Chem. Soc. 1999, 121, 8475-8478.
G. Bioorg. Med. Chem. Lett. 2002, 12, 701-704.
(30) Xu, Y.; Qian, L.; Pontsler, A. V.; McIntyre, T. M.; Prestwich, G.
(34) Blaszczak, L. C.; McMurry, J. E. J. Org. Chem. 1974, 39, 258-
D. Tetrahedron 2004, 60, 43-49.
31) Peleg, S.; Petersen, K. S.; Suh, B. C.; Dolan, P.; Agoston, E. S.;
Kensler, T. W.; Posner, G. H. J. Med. Chem. 2006, 49, 7513-7517.
32) Erickson, J. A.; McLoughlin, J. I. J. Org. Chem. 1995, 60, 1626-
631.
259.
(
(35) Asano, Y.; Yamada, A.; Kato, Y.; Yamaguchi, K.; Hibino, Y.; Hirai,
K.; Kondo, K. J. Org. Chem. 1990, 55, 5567-5571.
(36) Krix, G.; Bommarius, A. S.; Drauz, K.; Kottenhahn, M.; Schwarm,
M.; Kula, M.-R. J. Biotechnol. 1997, 53, 29-39.
(
1
5518
Org. Lett., Vol. 9, No. 26, 2007

higher ylide concentrations produced the side product along
with the desired product. Accordingly, compound 1b was
synthesized using these optimized conditions.
meric excess (Figure 3A), showing unprecedented tolerance
to the polar CF H functionality. The high polarity of Qfl is
2
Compound 1a was then subjected to hydrogenation condi-
12
tions to reduce the alkene bond as described in the literature.
However, we obtained either unreacted reactant 1a or the
over hydrogenated alcohol (6, Figure 2) in the presence of
the desired product 2a (Table 1, entries 1 and 2). Changing
Table 1. Reduction of Fluorine-Containing Unsaturated
Pyruvate Esters
product ratioa
time (h) reactant:desired:side
Figure 3. HPLC chromatograms for dl-Qfl obtained from racemic
synthesis and Qfl from enzymatic reductive amination as eluted
off a CrownPak CR(+) column (Panel A) and for peptides KdlQfl
and KQfl synthesized from dl-Qfl and Qfl, recpectively, as eluted
off a C18 reverse phase column (Panel B).
entry
R
conditions
1
2
3
4
5
6
CF3
CF3
CF3
10 psi H2, Pd/C
10 psi H2, Pd/C
TiCl3(aq)
0.5
1.5
0.25
0.5
8.0
15:79:6
0:79:21
2:98:0
22:39:39
26:53:21
0:93:7
CF2H TiCl3(aq)
CF2H TiCl3(aq)
CF2H 10 psi H2, Pd/C
reflected in the short retention time (t
reverse phase high performance liquid chromatography
HPLC) column (Table 2). Based on the t , the hydrophobic-
R
) as eluted off a C18
0.75
a
Determined by GC-MS.
(
R
2
the reaction time or H pressure did not improve the exclu-
R
Table 2. Reverse Phase HPLC Retention Time (t ), Helix
Propensity (w), and Helix Forming Energetics (∆G) for Qfl, Hfl,
and Leu
sive production of the desired product 2a. Interestingly,
McMurry and co-workers have reported the selective reduc-
tion of alkene groups in enedicarbonyl compounds using
(aq).34 This reaction seemed particularly suitable for
amino acid
tR (min)a
w
∆G (kcal‚mol )
-1 b
TiCl
3
selectively reducing electron-deficient alkenes, such as
those in compounds 1a and 1b due to the presence of
multiple electron-withdrawing fluorines and the conjugated
carbonyl. Compound 1a was selectively reduced to 2a
Qfl
Hfl
Leu
4.8
13.3
8.4
0.445 ( 0.052
0.128 ( 0.023
1.06 ( 0.12
0.440 ( 0.068
1.12 ( 0.11
-0.0317 ( 0.0654
a
Retention time as eluted off an analytical C18 reverse phase column.
by using TiCl
However, reducing the number of fluorines from 6 (R )
CF ) to 4 (R ) CF H) resulted in significantly lower yields
Table 1, entries 4 and 5), showing that TiCl (aq) is only
3
(aq) nearly quantitatively (Table 1, entry 3).
b ∆G ) -RT‚ln(w).
3
2
(
3
ity of the Leu analogues followed the trend Hfl > Leu >
Qfl. The S configuration for the CR center was confirmed
based on enantiomeric preference of phenylalanine dehy-
suitable for selectively reducing highly electron-deficient
alkenes. As such, we performed hydrogenation on com-
pound 1b to give the desired product 2b (Table 1, entry 6)
instead.
3
5,36
drogenase,
convention of the elution profile for the
37
CrownPak CR(+) column, and the Clough-Lutz-Jirgen-
son rule on how specific rotation changes with pH.38 The
backbone nitrogen was protected with Fmoc (9-fluorenyl-
methyloxycarbonyl) to afford the appropriately protected
amino acid for solid-phase peptide synthesis. Importantly,
all transformations including the enzymatic reductive ami-
nation were used to synthesize the amino acids on gram scale.
The sequence of peptide KQfl, Ac-YGGKAAAA-KAX-
Upon hydrolyzing compound 2a, reductive amination to
give Hfl (3a) was performed with NADH mediated by
phenylalanine dehydrogenase,1
5,35,36
which is known to
operate on a variety of hydrophobic hydrocarbon side
chains differing in size. The reductive amination proceeds
through in situ formation of an imine between the R-keto
acid and the ammonium in the buffer, followed by enanti-
oselective imine reduction with NADH mediated by pheny-
lalanine dehydrogenase. Since quantitative consumption
of NADH would not be economical, formate hydrogenase
AAKAAAAK-NH
2
(X ) Qfl), was designed based on
peptides studied by Baldwin for measuring helix propen-
1
5,39
sity.
The peptide KQfl was synthesized by standard
was added to regenerate NADH with concomitant sacrificial
protocols for solid-phase peptide synthesis (SPPS), using
Fmoc-based chemistry. The identity of the peptide was
oxidation of the formate in the buffer.1
5,35,36
Surprisingly,
40
phenylalanine dehydrogenase also converted the polar
tetrafluoroleucine pyruvate into Qfl (3b) with 98% enantio-
confirmed by matrix-assisted laser desorption ionization time-
of-flight mass spectrometry (MALDI-TOF), despite concerns
Org. Lett., Vol. 9, No. 26, 2007
5519

regarding repeated use of piperidine for Fmoc removal during
peptide synthesis in the presence of the acidic protons on
the CF H groups. Also, we independently synthesized and
2
incorporated the racemic dl-Qfl to give peptide KdlQfl to
check for racemization of the CR center during peptide
synthesis. Based on C18 reverse phase HPLC chromatograms
formation energetics. Apparently, the high polarity of the
Qfl side chain would avoid the energetic penalty for side
chain burial in the unfolded state as speculated for Hfl,15
but would not provide shielding of the helix hydrogen bond
2
0
from water like Leu. Nevertheless, the helix propensity of
Qfl is the highest among all fluoro-amino acids measured
1
5
(Figure 3B), peptide KdlQfl exhibited two peaks, both of
to date.
which gave the same m/z by MALDI-TOF as peptide KQfl.
Furthermore, the retention time of the trailing peak matched
that for peptide KQfl. Importantly, Qfl was incorporated into
peptides by SPPS without any protecting group, and the
stereochemistry of the CR center remained intact in the
process.
We have developed a short and facile stereoselective
chemoenzymatic synthesis for both Hfl and Qfl on gram
scale. TiCl (aq) was used to selectively reduce a highly
3
electron-deficient alkene for the synthesis of Hfl. Phenyla-
lanine dehydrogenase is surprisingly capable of producing
the polar Qfl despite the inherent preference for hydrophobic
3
5,36
Peptide KQfl was monomeric in solution based on
sedimentation equilibrium experiments by analytical ultra-
substrates.
This result may lead to further utility of this
enzyme in synthesizing amino acids with polar side chains
as well as hydrophobic ones. Furthermore, Qfl can be readily
incorporated into peptides by standard protocols for solid-
phase peptide synthesis without any side chain protection.
Importantly, the helix formation energetics of Qfl is not only
4
1
centrifugation. Therefore, circular dichroism (CD) spectra
of KQfl should accurately reflect the helical content in the
absence of any intermolecular interactions. The CD spectra
of KQfl showed helical content between peptides KLeu and
KHfl (Figure 4). The helix propensity (w) was calculated
-
1
more favorable compared to Hfl by 0.68 kcal‚mol , but is
the most favorable for all fluoro-amino acids measured to
1
5
date. The availability, ease of incorporation, and favorable
helix propensity of Qfl sets the stage for future studies to
probe if the dipole moment and hydrogen bonds involving
the fluorines are important factors in the fluoro-stabilization
-1
-1 7,15
effect of more than 1.5 kcal‚mol ‚residue . Furthermore,
Qfl expands the repertoire of R-amino acids for peptide/
protein design, and provides a facile means to introduce
2
CF H groups for developing bioactive compounds.
Acknowledgment. This work was supported by the
NYSTAR James D. Watson Investigator Program, American
Chemical Society Petroleum Research Fund (PRF No.
4
4532-G4), Kapoor funds, and the State University of New
York at Buffalo. The authors would like to thank Dr. Robert
Fairman and Bashkim Kokona at Haverford College for
performing the sedimentation equilibrium experiments.
Figure 4. Circular dichroism spectra of peptides KQfl, KHfl, and
KLeu at pH 7 (273 K) in 1 mM phosphate, borate, and citrate with
Supporting Information Available: Experimental details
for the synthesis and characterization of tetrafluoroleucine
and peptide KQfl, and experimental methods for circular
dichroism spectroscopy and calculating the helix propensity
of Qfl. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
M NaCl.
from the CD data by using modified Lifson-Roig theory15,42,43
Table 2). Although the helix propensity of Qfl is half of
(
that for Leu, the helix-forming energetics for Qfl is more
favorable than that for Hfl by 0.68 kcal‚mol-1 (Table 2).
The favorable helix propensity for Leu can be attributed to
the shielding of the helix hydrogen bond from water by the
hydrophobic side chain,20 resulting in a more stable helix
hydrogen bond and thus more stable helix conformation. The
low helix propensity of Hfl may be due to partial or full
burial of the hydrophobic fluorocarbon side chain in the un-
folded state with more exposure of this hydrophobic side
chain in the helix state,15 leading to unfavorable helix
OL702470J
(37) Hilton, M.; Armstrong, D. W. J. Liq. Chromatogr. 1991, 14, 9-28.
(38) Greenstein, J. P.; Winitz, W. D. In Chemistry of the Amino Acids;
John Wiley and Sons: New York, 1961; pp 1879-2155.
(39) Chakrabartty, A.; Kortemme, T.; Baldwin, R. L. Protein Sci. 1994,
3
, 843-852.
40) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-
14.
(41) See the Supporting Information.
42) Doig, A. J.; Chakrabartty, A.; Klingler, T. M.; Baldwin, R. L.
(
2
(
Biochemistry 1994, 33, 3396-3403.
(43) Andersen, N. H.; Tong, H. Protein Sci. 1997, 6, 1920-1936.
5520
Org. Lett., Vol. 9, No. 26, 2007