Efficient synthesis of suitably protected β-difluoroalanine and γ-difluorothreonine from L-ascorbic acid

Article abstract of DOI:10.1021/ol062401a

(Chemical Equation Presented) Fluorinated amino acids are useful building blocks for the preparation of biologically active peptides and peptidomimetics with increased metabolic stability. We report here the synthesis of two fluorinated amino acids, β-dif

Full text of DOI:10.1021/ol062401a

ORGANIC
LETTERS
2007
Vol. 9, No. 1
41-44
Efficient Synthesis of Suitably Protected
â
γ
-Difluoroalanine and
-Difluorothreonine from -Ascorbic
L
Acid
Gongyong Li and Wilfred A. van der Donk*
Roger Adams Laboratory, Department of Chemistry, UniVersity of Illinois at Urbanas
Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801
Vddonk@scs.uiuc.edu
Received September 29, 2006
ABSTRACT
Fluorinated amino acids are useful building blocks for the preparation of biologically active peptides and peptidomimetics with increased
metabolic stability. We report here the synthesis of two fluorinated amino acids, -difluoroalanine and -difluorothreonine, as analogues of
â
γ
Ser and Thr, respectively. These compounds were suitably protected for Fmoc-based solid-phase peptide synthesis. Once incorporated into
peptides, they may serve as alternative substrates or inhibitors of lantibiotic synthetases that posttranslationally dehydrate Ser and Thr
residues to dehydroalanine and dehydrobutyrine, respectively.
Michael acceptors have been popular functionalities for the
design of enzyme inhibitors and active site affinity labels.1
Dehydroalanines (Dha) and dehydrobutyrines (Dhb) are
potential Michael acceptors and are present in a large number
of natural products, including the microcystins,2 nodularin,3
thiostrepton and other thiopeptides,4 and the lantibiotics.5 The
electrophilicity of dehydroalanine (Dha) is significantly
moderated compared to acrylamides due to the inherent
enamine functionality in dehydroamino acids. This may
explain why natural products containing dehydroalanines
often interact with their targets by noncovalent mechanisms6
despite the presence of the Michael acceptor. Increasing the
electrophilicity of dehydroalanines in natural products or
designed inhibitors may lead to the development of powerful
tools for mechanistic enzymology or for use in cell biology
and signal transduction. Introduction of an electron-
withdrawing group on the terminal vinyl carbon would
provide for the desired increased reactivity. If this functional-
ity also consisted of a good leaving group, the Michael
addition could be rendered irreversible by way of an
addition-elimination pathway (Scheme 1).7 Fluorine-
substituted dehydroalanines would be particularly attractive
because of the small steric requirements of the fluorine
substituent. Moreover, fluorinated amino acids have received
(1) (a) Walsh, C. Tetrahedron 1982, 38, 871. (b) Walsh, C. T. Annu.
ReV. Biochem. 1984, 53, 493. (c) Silverman, R. B. Mechanism-based Enzyme
InactiVation: Chemistry and Enzymology; CRC Press: Boca Raton, FL,
1988.
(2) (a) Botes, D.; Tuinman, A.; Wessels, P.; Viljoen, C.; Kruger, H.;
Williams, D. H.; Santikarn, S.; Smith, R.; Hammond, S. J. Chem. Soc.,
Perkin Trans. 1 1984, 2311. (b) Painuly, P.; Perez, R.; Fukai, T.; Shimizu,
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953.
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1 1985, 2747. (c) Rinehart, K. L.; Harada, K.; Namikoshi, M.; Chen, C.;
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A.; Carmicheal, W. J. Am. Chem. Soc. 1988, 110, 8557.
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225, 233. (b) Walker, J.; Olesker, A.; Valente, L.; Rabanal, R.; Lukacs, G.
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10.1021/ol062401a CCC: $37.00
© 2007 American Chemical Society
Published on Web 12/13/2006

Scheme 1
Scheme 2
much attention due to their potential applications in
medicine.8-10
In previous studies, we have developed a synthetic
methodology to prepare fluorinated dehydroalanines.11 In this
contribution, we describe the synthesis of precursor amino
acids that are envisioned to be potential substrates for
lantibiotic synthetases. This class of enzymes catalyzes the
dehydration of Ser and Thr residues in their substrate peptides
resulting in formation of Dha and Dhb structures.5 With the
recent successful in vitro reconstitution of these proteins,12
they can now be evaluated as potential tools for the
construction of more reactive Dha and Dhb analogues. One
possibility would be the replacement of a Ser in the substrates
for dehydration by a difluoroalanine. Enzymatic elimination
of one of the fluorines of difluoroalanine11b would then
produce a fluoro-Dha (Scheme 2). Alternatively, replacement
of the methyl group of Thr with a fluorinated methyl group
and subsequent enzymatic dehydration would result in a Dhb
analogue with increased electrophilicity at the â-carbon.
We report here efficient routes to (2R)-â-difluoroalanine
(1) and (2S,3S)-γ-difluorothreonine (2) appropriately pro-
tected for use in Fmoc-based solid-phase peptide synthesis
(SPPS). We elected to prepare difluorinated alanine and
threonine derivatives as they would be sterically less
demanding than the corresponding trifluorinated analogues
and because trifluoroalanine incorporation into peptides is
challenging and the products have been reported to have low
chemical and configurational stability at physiological pH.13
Previous syntheses of difluoroalanine derivatives have been
mostly racemic14 with some asymmetric routes reported.15
Synthesis of Fmoc-â-difluoroalanine (1). L-Glyceralde-
hyde acetonide 3 is commercially available or can be readily
prepared from L-ascorbic acid in a large scale in 47% overall
yield16 or from commercially available 5,6-isopropylidene-
L-gulono-1,4-lactone in one step (56%).17 The compound was
fluorinated with diethylamino sulfurtrifluoride (DAST) to
afford compound 4 in 89% yield (Scheme 3).18 Treatment
of 4 with hydrochloric acid in methanol was followed by
selective protection of the primary hydroxyl with a tert-
butyldimethylsilyl group in 88% yield. The secondary alcohol
in product 5 was transformed into azide 6 in 70% yield by
reaction with trifluoromethylsulfonic anhydride in pyridine
and subsequent treatment with sodium azide. The azide 6
was transformed in 86% yield into Fmoc-protected amine 7
by reduction of the azide group to the amine and reaction
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42
Org. Lett., Vol. 9, No. 1, 2007

Scheme 3
Scheme 4
with FmocCl/NaHCO3.19 The TBS group was removed with
HF/pyridine to afford Fmoc-protected amino alcohol in
quantitative yield. Attempts to oxidize the amino alcohol by
NaIO4/RuCl3 induced partial Fmoc deprotection resulting in
isolation of the desired amino acid 1 in only 33% yield.
However, oxidation of the amino alcohol with the Jones
reagent afforded the target 1 in 81% yield without loss of
stereochemical purity.19 Using L-glyceraldehyde acetonide
(3) as the starting material, Fmoc-difluoro-Ala-OH (1) was
obtained in an overall yield of 38%.
hydrogenated in the presence of Pd(OH)2/C to afford the
corresponding amino alcohol, which was reacted with
FmocCl/NaHCO3 to afford compound 13 in 82% yield. It
should be noted that using Pd/C as the catalyst the azide
group in compound 12 could be selectively reduced without
debenzylation. Attempts to protect the hydroxyl group of
compound 13 with isobutene and catalytic H2SO4 provided
the desired product in only 21% yield with products in which
the TBS group had been removed making up the mass
balance. However, repeated treatment of compound 13 with
t-BuBr/Ag2O provided 14 in an overall yield of 81%.
Deprotection of the TBS group was carried out in quantitative
yield by using a solution of commercial HF/pyridine in THF
after adjustment of the pH to 5 by the addition of pyridine;
without the addition of pyridine, a complex product mixture
was obtained. Finally, subjection of the crude amino alcohol
to the Jones reagent afforded the final product Fmoc-γ-
difluoro-Thr(OtBu)-OH (2) in 73% yield.
Synthesis of Fmoc-γ-difluoro-Thr(OtBu)-OH (2). Given
the successful synthesis of Fmoc-â-difluoro-Ala-OH (1) from
L-ascorbic acid, we attempted to use the same strategy to
prepare Fmoc-γ-difluoro-Thr(OtBu)-OH20 from advanced
intermediate 8, prepared from L-ascorbic acid in three steps
in 76% yield.21 The hydroxyl group of compound 8 was
protected with either a benzyl or tert-butyl group to give
compounds 9a and 9b in 91% and 90% yields, respectively.
The benzyl protection group provides more versatility
downstream, whereas the tert-butyl group needs to be
installed eventually in the final product because it is the
protecting group of choice for alcohols in Fmoc-based SPPS.
Compound 9a was reduced by DIBAL-H and fluorinated
with DAST to yield difluoride 10 in 67% yield. Only one
In summary, two difluoroamino acids were efficiently
synthesized from L-ascorbic acid. Given the well-known
success of fluorine-containing pharmaceuticals,22 including
fluoropeptides,23 the current approach expands the arsenal
of fluorinated moieties that can be used in the preparation
of bioactive molecules. The fluorinated amino acids will be
incorporated into the peptide substrate for lacticin 481
synthetase12a to investigate the enzymatic generation of
fluorinated dehydroamino acids within oligopeptides.
1
diastereomer was detected by H NMR analysis. However,
treatment of 9b did not provide compound 10b, presumably
because of steric hindrance. The isopropylidene group was
then removed from 10 with hydrochloric acid in methanol,
and the liberated primary alcohol was selectively protected
with a tert-butyldimethylsilyl group to afford compound 11
in 86% yield (Scheme 4).
The secondary alcohol of compound 11 was converted to
the trifluoromethylsulfonate, and the crude product was
subsequently reacted with sodium azide providing compound
12 in a yield of 79%. The azide functionality in 12 was
Acknowledgment. This work was supported by the
National Institutes of Health (GM58822). W.A.V. is an
(19) Mosher’s amide analysis of the free amines 7a and 1a both showed
an er of 1.00:0.04. See Supporting Information.
(20) Both enantiomers of γ-difluoro Thr have been previously prepared
in unprotected form using lipase-catalyzed resolutions: Shimizu, M.;
Yokota, T.; Fujimori, K.; Fujisawa, T. Tetrahedron: Asymmetry 1993, 4,
835.
(22) (a) Abeles, R. H.; Alston, T. A. J. Biol. Chem. 1990, 265, 16705-
16708. (b) Filler, R.; Kobayashi, Y.; Yagupolskii, L. M. Organofluorine
Compounds in Medicinal Chemistry and Biomedical Applications; Elsevi-
er: New York, 1993. (c) Ojima, I.; Welch, J. T.; McCarthy, J. R. Biomedical
Frontiers of Fluorine Chemistry; American Chemical Society: Washington
DC, 1996.
(21) Dahlgren, A.; Kvarnstro¨m, I.; Vrang, L.; Hamelink, E.; Hallberg,
A.; Rosenquist, A.; Samuelsson, B. Bioorg. Med. Chem. 2003, 11, 827.
(23) Imperiali, B. AdV. Biotechnol. Processes 1988, 10, 97-129.
Org. Lett., Vol. 9, No. 1, 2007
43

Alfred P. Sloan Fellow and Camille Dreyfus Teacher-
Scholar. NMR spectra were obtained in the Varian Oxford
Instrument Center for Excellence, funded in part by the W.
M. Keck Foundation, NIH (S10 RR10444), and NSF (CHE
96-10502).
Supporting Information Available: Detailed experi-
1
mental procedures and copies of H, 13C, and 19F NMR
spectra of all unknown compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL062401A
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Org. Lett., Vol. 9, No. 1, 2007