Practical syntheses of 4-fluoroprolines

Article abstract of DOI:10.1016/j.jfluchem.2008.06.024

4-Fluoroprolines are among the most useful nonnatural amino acids in chemical biology. Here, practical routes are reported for the synthesis of the 2S,4R, 2S,4S, and 2R,4S diastereomers of 4-fluoroproline. Each route starts with (2S,4R)-4-hydroxyproline, which is a prevalent component of collagen and hence readily available, and uses a fluoride salt to install the fluoro group. Hence, the routes provide process-scale access to these useful nonnatural amino acids.

Full text of DOI:10.1016/j.jfluchem.2008.06.024

Journal of Fluorine Chemistry 129 (2008) 781–784
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Practical syntheses of 4-fluoroprolines
b
b
b
Mukund S. Chorghade a, , Debendra K. Mohapatra , Gokarneswar Sahoo , Mukund K. Gurjar ,
*
Manish V. Mandlecha c, Nitin Bhoite c, Santosh Moghe c, Ronald T. Raines
d,**
a THINQ Pharma, Natick, MA 01760, USA
b National Chemical Laboratory, Pune, India
c THINQ Pharma–CRO, Mumbai, India
d Departments of Biochemistry and Chemistry, University of Wisconsin–Madison, Madison, WI 53706, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
4-Fluoroprolines are among the most useful nonnatural amino acids in chemical biology. Here, practical
routes are reported for the synthesis of the 2S,4R, 2S,4S, and 2R,4S diastereomers of 4-fluoroproline. Each
route starts with (2S,4R)-4-hydroxyproline, which is a prevalent component of collagen and hence
readily available, and uses a fluoride salt to install the fluoro group. Hence, the routes provide process-
scale access to these useful nonnatural amino acids.
Received 6 May 2008
Received in revised form 20 June 2008
Accepted 22 June 2008
Available online 2 July 2008
ß 2008 Elsevier B.V. All rights reserved.
Keywords:
Collagen
4-Fluoroproline
4-Hydroxyproline
Nonnatural amino acid
Process-scale synthesis
1. Introduction
The chemical synthesis of a 4-fluoroproline often begins
with a 4-hydroxyproline [9]. (2S,4R)-4-Hydroxyproline (HypOH),
The ability to incorporate nonnatural amino acids into proteins is
altering the landscape in chemical biology and related fields [1].
Perhaps no other nonnatural amino acid has been as useful in
enhancing the conformational stability of proteins as have 4-
fluoroprolines. In the proper context, the 4R and 4S diastereomers of
4-fluoroproline have been shown to increase dramatically the
conformational stability of collagen [2], which is the most abundant
protein in animals. In addition, 4-fluoroproline residues can endow
conformational stability upon other proteins [3] as well as peptides
[4]. This enhanced stability is the result of the increased tendency of
4-fluoroproline residues to adopt a C -exo or C -endo pyrrolidine
ring pucker (2S,4R or 2S,4S diastereomer, respectively), or form a
trans or cis peptide bond (2S,4R or 2S,4S diastereomer, respectively).
These conformational preferences arise from two stereoelectronic
effects [5]: a gauche effect that fixes the pyrrolidine ring pucker [6,7],
and ann ! p* interactionthatstabilizesthe trans peptidebond[6,8].
Because of these desirable attributes, there is a growing need to
access 4-fluoroprolines.
which is a prevalent component of collagen, is an especially
advantageous starting material for the synthesis of 4-fluoropro-
lines. The prevalence of this amino acid is extraordinary: the
abundance of Hyp within animal proteins is ꢀ4%, a value cal-
culated from the abundance of collagen amongst animal proteins
(1/3) and the prevalence of Hyp within collagen (ꢀ38% ꢁ 1/
3) [10]. Thus, the abundance of Hyp in animals exceeds that of
seven ‘‘common’’ amino acids: Cys, Gln, His, Met, Phe, Trp, and Tyr
[11].
Typically, thekeyreactioninsyntheticroutes toa4-fluoroproline
involves the SN2 displacement of an activated hydroxyl group in a 4-
hydroxyproline by a fluoride ion, resulting in the formation of a C–F
bond with the inversion of configuration at C-4. (Diethylamino)-
Sulfur trifluoride (DAST [12]) or a congener, such as 4-morpholi-
nosulfur trifluoride (morph-DAST), is typically employed in this
strategy [9,13], as such reagents can both activate the hydroxyl
group and supply the fluoride ion. DAST and its congeners are,
however, both expensive and explosive [14]. Moreover, the
analogous precursor of (2S,4R)-4-fluoroproline – (2S,4S)-4-hydro-
xyproline – is itself a nonnatural amino acid that is expensive.
Here, we report on practical synthetic routes to three 4-
flouroproline diasteromers: 2S,4R (1), 2S,4S (2), and 2R,4S (3). The
routes all start with HypOH, and none use a DAST-like reagent. All
are amenable to a process scale.
g
g
* Corresponding author. Tel.: +1 508 651 7809; fax: +1 508 651 7920.
** Corresponding author. Tel.: +1 608 262 8588; fax: +1 608 262 3453.
E-mail addresses: chorghade@comcast.net (M.S. Chorghade),
rtraines@wisc.edu (R.T. Raines).
0022-1139/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2008.06.024

782
M.S. Chorghade et al. / Journal of Fluorine Chemistry 129 (2008) 781–784
Scheme 1.
2. Results and discussion
carbamate. Mesylation, followed by the formation of lactone and
its hydrolysis, inverted the stereochemistry at C-4. Compound 7
can be taken on to (2S,4R)-4-fluoroproline (1) by the same three
steps used to convert compound 4 to (2S,4S)-4-fluoroproline (2)
with similar yields (data not shown).
The 2S,4S diastereomer of 4-fluoroproline (2) was synthesized
from HypOH by using the SN2 reaction of a fluoride ion to invert the
configuration at C-4. Specifically, (2S,4S)-4-fluoroproline (2) was
synthesized in four steps from HypOH with an overall yield of 16%.
As shown in Scheme 1, the hydroxyl group of compound 4 was
activatedwithtrifluoromethanesulfonicanhydride,andtheensuing
triflate was displaced with tetra-n-butyl ammonium fluoride (TBAF
[15]) to yield protected (2S,4S)-4-fluoroproline; deprotection with
acid gave the 2S,4S diastereomer of 4-fluoroproline (2).
Likewise, key precursors to the 2S,4R and 2R,4S diastereomers
of 4-fluoroproline (1 and 3) were synthesized from HypOH. The
routes to these precursors have a notable feature. The route to the
2S,4R diastereomer epimerizes C-4; the route to the 2R,4S
diastereomer epimerizes C-2. Each of these epimerizations was
accomplished with a distinct lactonization reaction.
(2R,4R)-4-Hydroxyproline (10) was synthesized from HypOH in
one step with a yield of 50%. As shown in Scheme 2, HypOH was
treated with acetic anhydride to produce (2R,4R)-4-hydroxypro-
line lactone. This remarkable transformation has been proposed to
proceed via a bicyclic mesoionic intermediate [16], and was used
recently in the synthesis of alkaloids from HypOH [17]. The
reaction conditions racemize C-2 of other amino acids, but lead to a
single enantiomer here because lactonization traps the 2R epimer.
The lactone was hydrolyzed in acid to yield (2R,4R)-4-hydroxypro-
line. Compound 10 can be taken on to (2R,4S)-4-fluoroproline (3)
by the same four steps used to convert HypOH to (2S,4S)-4-
fluoroproline (2) with similar yields (data not shown).
N-Boc-(2S,4S)-4-hydroxyproline (7) was synthesized in four
steps from HypOH with an overall yield of 72%. As shown in
Scheme 1, the amino group of HypOH was protected as a Boc
3. Conclusion
We have described practical routes to the 2S,4R, 2S,4S, and 2R,4R
diastereomers of 4-fluoroproline. These routes, which we have used
to synthesize 4-fluoroprolines on a kilogram scale, provide ready
access to a nonnatural amino acid of growing importance to
chemical biology. Finally, we note that these routes accommodate
the large-scale synthesis of [F-18]4-fluoroprolines. These probes,
along with positron emission tomography, have proven to be useful
for monitoring the healing of wounds, permeability of the blood–
brain barrier, and other physiological processes [18].
4. Experimental
4.1. Synthesis of N-Boc-(2S,4S)-4-hydroxyproline (7)
4.1.1. N-Boc-(2S,4R)-4-hydroxyproline (4)
HypOH (15.0 g, 0.114 mol) was dissolved in 150 mL of 2:1 THF/
H2O. To this solution were added 50 mL of 10% (w/v) NaOH(aq) and
Scheme 2.

M.S. Chorghade et al. / Journal of Fluorine Chemistry 129 (2008) 781–784
783
Boc anhydride (30.0 g, 0.137 mol), and the resulting solution was
stirred at room temperature overnight. The THF was removed by
rotary evaporation under reduced pressure. The residue was
dissolved in ethyl acetate, and the pH was adjusted to 2 by the
addition of 10% (w/v) KHSO4(aq). The acidic solution was extracted
several times with ethyl acetate. The combined organic extracts
were washed with H2O and brine, and then dried over anhydrous
Na2SO4(s). The desiccant was removed by filtration, and the
solvent was removed by rotary evaporation under reduced
pressure to give 4 (24.7 g, 92%) as a syrup.
in 100 mL of THF and cooled to 4 8C. To this solution was added a
1.0 M solution of TBAF in THF (1.15 g, 2.5 eq). The resulting
mixture was stirred at 4 8C overnight, and then concentrated under
reduced pressure. Chromatography (silica gel, 1:1 ethyl acetate/
petroleum ether) furnished 9 (1.21 g, 40%) as a solid. (The
characterization of compound 9 was as described by Chiba et al.
[22]).
4.2.3. (2S,4S)-4-Fluoroproline (2)
Compound 9 (2.00 g, 8.09 mmol) was dissolved in 10 mL of 2N
HCl. The resulting solution was heated under reflux for 2–4 h. After
the reaction was complete (as monitored by TLC), the solution was
decolorized with charcoal while still hot, and filtered through a pad
of Celite1. Water was removed by rotary evaporation under
reduced pressure and as an azeotrope with dry toluene. Crystal-
lization (1:1 ethyl acetate/petroleum ether) afforded the hydro-
chloride salt of 2 (0.67 g, 49%) as a white solid.
4.1.2. N-Boc-(2S,4R)-4-mesylproline (5)
Compound 4 (2.00 g, 8.65 mmol) was dissolved in CH2Cl2 at
0 8C. Pyridine (1.40 mL, 17.3 mmol) and mesyl chloride (1.33 mL,
17.3 mmol) were added, and the resulting solution was stirred
overnight. The reaction was quenched with water, and the aqueous
layer was extracted with CH2Cl2 (3 ꢁ 50 mL). The combined
extracts were washed with water and brine, dried over anhydrous
Na2SO4(s), and evaporated to dryness. The crude product 5 was
taken on without purification. (The characterization of compound
5 was as described by Scha¨fer and coworkers [19].)
4.3. Synthesis of (2S,4S)-4-hydroxyproline (10)
A stirred mixture of HypOH (6.55 g, 50.0 mmol) in 40 mL of
acetic anhydride was heated at 90 8C for 7 h under N2(g). Solvent
was removed under reduced pressure. The thick oil thus obtained
was dissolved in 25 mL of 2N HCl, and the resulting solution was
heated at reflux for 3 h. The pH of the solution was then adjusted to
6 by the addition of NaOH(aq), water was removed under reduced
pressure, and the product was isolated and purified by crystal-
lization (1:1 ethyl acetate/petroleum ether) to yield 10 (3.27 g,
50%) as a solid. (The characterization of compound 10 was as
described by Dalla Croce and La Rosa [16].)
4.1.3. N-Boc-(2S,4S)-4-hydroxyproline lactone (6)
Compound 5 (2.80 g, 9.09 mmol) was dissolved in THF contain-
ing KOtBu (1.22 g, 10.9 mmol) at 0 8C. The reaction mixture was
stirred at room temperature for 12 h. After the reaction was
complete (asmonitored by TLC), the reaction mixture was quenched
with water and extracted with ethyl acetate (2 ꢁ 50 mL) to furnish 6
(1.46 g, 78% over two steps) as a syrup. (The characterization of
compound 6 was as described by Silverman and coworkers [20].)
4.1.4. N-Boc-(2S,4S)-4-hydroxyproline (7)
Acknowledgements
Compound 6 (1.00 g, 4.69 mmol) was dissolved in 35 mL of
2:2:3 THF/MeOH/H2O. LiOH (0.33 g, 14.1 mmol) was added, and
the resulting solution was stirred overnight. The organic solvent
was removed by rotary evaporation under reduced pressure. The
residue thus obtained was dissolved in 25 mL of ethyl acetate, and
the resulting solution was acidified with a saturated aqueous
solution of KHSO4. The aqueous layer was extracted with ethyl
acetate (2 ꢁ 50 mL), and the combined organic extracts were dried
over anhydrous Na2SO4(s) and concentrated under reduced
pressure to yield 7 (1.09 g, quant) as a syrup.
We are grateful to A. Choudhary and M.D. Shoulders for
contributive discussions. This work was supported in part by grant
AR044276 (NIH).
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