Synthesis and Conformational Properties of 3,4-Difluoro- l -prolines

Article abstract of DOI:10.1021/acs.joc.8b02920

Fluorinated proline derivatives have found diverse applications in areas ranging from medicinal chemistry over structural biochemistry to organocatalysis. Depending on the stereochemistry of monofluorination at the proline 3- or 4-position, different effe

Full text of DOI:10.1021/acs.joc.8b02920

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Article
Synthesis and conformational properties of 3,4-difluoro-L-prolines
Gert-Jan Hofman, Emile Ottoy, Mark E. Light, Bruno Kieffer,
Jose C. Martins, Ilya Kuprov, Davy Sinnaeve, and Bruno Linclau
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02920 • Publication Date (Web): 19 Feb 2019
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Synthesis and conformational properties of 3,4-
difluoro-L-prolines
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a,b
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Gert-Jan Hofman, Emile Ottoy, Mark E. Light, Bruno Kieffer, Jose C. Martins, Ilya
Kuprov, Davy Sinnaeve and Bruno Linclaua,*
a
b,
a
Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, United
Kingdom.
b
Department of Organic and Macromolecular Chemistry, Ghent University, Campus Sterre,
S4, Krijgslaan 281, B-9000 Ghent, Belgium.
c
Biomolecular NMR, University of Strasbourg, IGBMC-CNRS UMR 7104 – INSERM
U1258, 1 rue Laurent Fries / BP 10142 / 67404 Illkirch CEDEX/France.
bruno.linclau@soton.ac.uk

Current address: Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576,
Université de Lille, 59655 Villeneuve d’Ascq, France
RECEIVED DATE
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pucker stabilization:
pucker stabilization:
endo
F
F
exo
O
O
endo
exo
N
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F
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OMe
OMe
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exo
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“
pucker-neutral”
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OMe
O
Abstract—Fluorinated proline derivatives have found diverse applications in areas ranging
from medicinal chemistry over structural biochemistry to organocatalysis. Depending on the
stereochemistry of monofluorination at the proline 3- or 4-position, different effects on the
conformational properties of proline (ring pucker, cis/trans isomerization) are introduced. With
fluorination at both 3- and 4-positions, matching or mismatching effects can occur depending
on the relative stereochemistry. Here we report, in full, the syntheses and conformational
properties of three out of the four possible 3,4-difluoro-L-proline diastereoisomers. The yet
unreported conformational properties are described for (3S,4S)- and (3R,4R)-difluoro-L-
proline, which are shown to bias ring pucker and cis-trans ratios on the same order of
magnitude as their respective monofluorinated progenitors, although with significantly faster
amide cis-trans isomerization rates. The reported analogues thus expand the scope of available
fluorinated proline analogues as tools to tailor proline’s distinct conformational and dynamical
properties, allowing to interrogate its role in, for instance, protein stability or folding.
INTRODUCTION
Fluorination of organic molecules has proven to be a highly useful tool to manipulate their
conformational and electronic properties with minimal steric effects.1-7 Fluorination of the L-
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proline ring has been heavily exploited for conformational control of its ring pucker. For


example, the five-membered proline ring conformation can be biased to either a C exo or a C
endo pucker by introducing a (4R)-fluoro group (1, Figure 1) or a (4S)-fluoro group (2),
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respectively, an effect attributed to σ →σ
hyperconjugation interactions. Besides ring
CF
CH
pucker, fluorination also strongly influences the cis/trans ratio of the Xaa-Pro peptide bond
1
0
relative to proline in a solvent-dependent way. The inductive effect of fluorine reduces the
capacity for the nitrogen lone pair to conjugate with the amide carbonyl group, and thus to
contribute to the double bond character of the amide bond. As a consequence, the rotational
energy barrier is decreased and accelerated cis/trans isomerization is observed.11-13 The same
effect renders fluorinated prolines less basic,11,13,14 and the carboxylic acid group more acidic.15
a)
b)
F
(R)
O
N
O
H
CH⟶*CF

C exo pucker,
N
strong n⟶* interaction,
trans isomer more favored
compared to proline
O
O
F
(4R)-FPro 1
F
(S)
O
F
O
CH⟶*

C endo pucker,
CF
N
weak n⟶* interaction,
trans isomer less favored
compared to proline
N
O
O
H
(4S)-FPro 2
c)
O
O
n⟶* interaction stabilizes
the trans isomer
N
N
n⟶*
trans-Pro 3
O
O
cis-Pro 3


Figure 1 (a) (4R)-FPro 1 adopts an C exo pucker (b) (4S)-FPro 2 adopts an C endo pucker (c)
*
nπ interaction stabilizes the trans isomer
The combination of both conformational and dynamical effects make fluoroprolines valuable
tools for determining the significance of proline’s unique structural properties within proteins
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or peptides.8,14 Nevertheless, the first syntheses of (4R)-FPro 1 and (4S)-FPro 2 date back to
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965, although it took until the late nineties for this potential to be fully recognized. In a
landmark study investigating the mechanism behind collagen stability,9, 17-18 Raines and co-
workers applied fluoroprolines to revise the origins behind the extraordinary thermostability of
this protein, which forms triple helices out of Pro-Hyp-Gly repeats. Replacing (4R)-4-
hydroxyproline (Hyp) with (4R)-FPro 1 led to a more thermostable collagen mimic, which,
since fluorine is a weak hydrogen bond acceptor, disproved that a hydrogen bond network
involving the hydroxyl moiety of Hyp induces collagen stability. In contrast, replacing Hyp by
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(4S)-FPro 2 led to less stable collagen mimics. Since fluorine is more electronegative than a

hydroxyl group, (4R)-FPro favors the C exo pucker more strongly than Hyp, and because (4S)-


FPro favors the C endo pucker, this revealed that it is the strong preference for the C exo
pucker of Hyp that plays a key role for collagen stability. This ring pucker preorganizes the
*
dihedral angles in such a way that a favorable n→π interaction is promoted between the
carbonyl groups of two adjacent peptide bonds, favoring the trans amide bond rotamer.19
Interestingly, the ring pucker of the Pro residue preceding Hyp is also relevant for collagen
2
0
stability, which has equally been investigated using both 4- and 3-monofluorinated proline
variants.21
The case of collagen initiated many other demonstrations of the potential of proline fluorination
to investigate the distinct structural and dynamical properties of proline residues within
peptides and proteins, exploiting both the modulations of proline structure and cis-trans
8
isomerization kinetics. Indeed, modulating these properties by fluorination, rather than just
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fully eliminating them by mutating proline to non-proline residues, can provide a more
elegant approach towards uncovering the functional significance of proline’s unique properties.
1
9
Moreover, the introduction of fluorine allows the use of F NMR as a powerful means to
1
9
monitor residue-specific information. The exceptionally high responsivity of the F nucleus to
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changes in its (local) environment, in addition to the sparsity of the F spectrum, make F
NMR a very attractive means to monitor protein structural and dynamical changes, enzyme
catalysis, and ligand binding.23-28 Despite these clear advantages and earlier suggestions,29-31
to the best of our knowledge, there are only a very limited number of reports involving the full
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32,33
potential of F NMR in a fluoroproline peptide context.
However, if the FPro residue is to
1
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be used purely as a F NMR probe, the conformationally perturbing effects of fluorine must
be carefully considered. We recently introduced (3S,4R)-3,4-difluoroproline ((3S,4R)-FPro) 4
(
Figure 2) where the two fluorines have opposing pre-organizing effects, thus resulting in a
proline analogue with minimal conformational bias and minimal homonuclear coupling
1
9
34
complications for F NMR purposes.
Given the well-demonstrated importance of having fluoroprolines available with matching
conformational, kinetic and NMR properties for the application at hand, the synthesis of novel
3
5
fluorinated variants in optically pure form continues to be of interest. In addition, regardless


of whether such applications require conformationally neutral, C exo- or C endo-pucker
promoting fluoroprolines, the availability of more than one variant with similar conformational
1
9
properties, but well-separated F NMR chemical shifts, is of interest for site-specific multi-
residue labelling strategies of proteins, especially in the case of low-complexity sequences
found in proline-rich proteins such as collagen, but also many transcriptional activators. Hence,
we envisaged a convenient synthesis of the 3,4-difluoro-L-prolines 4-7 (Figure 2), in order to
expand the toolbox of proline analogues.
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R)
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(S)
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(S)
(
O
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OH
OH
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R)
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(S)
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N
H
N
H
OH
OH
6
7
Figure 2: Structures of the targeted 3,4-difluoroprolines
There exists only limited precedence for such difluorinated proline analogues (Scheme 1). A
Novartis patent describes the synthesis of N-Boc-7 in 14 steps from commercially available
3
,4-dehydroproline 8.36 After epoxidation and acid-catalyzed epoxide opening, the key
fluorination steps involve DAST-mediated deoxyfluorination reactions as shown in Scheme
1a. However, no yields or NMR data were reported. The second example (Scheme 1b) was
published by Fleet and coworkers, where deoxyfluorination of 16 using XtalFluor-
M/Et N•3HF did not lead to the desired difluorinated azetidine derivative (not shown), but
3
instead yielded the ring-expanded product 17.37 Deprotection of 17 led to (3R,4R)-3,4-
difluoroproline 6. Hence, in both cases, C–F bond introduction was achieved in sequential
fashion. Finally, our group recently reported a stereoselective synthesis of Boc-protected
(
3S,4R)-3,4-difluoroproline (N-Boc)-4, which featured a direct bis-deoxyfluorination step
3
4
(Scheme 1c). (3R,4S)-3,4-Dihydroxyproline 19a, obtained by selective dihydroxylation of
the corresponding 3,4-dehydroproline, was treated with nonafluorobutanesulfonyl fluoride
(
NfF) in combination with tetrabutylammonium triphenyldifluorosilicate (TBAT) to yield 20a
as the only observed 3,4-difluoroproline.
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O
F
OAll
F
OH
a) Novartis
O
O
OAc
1) DAST
F
(S)
F
(R)
OH
1) LiBH4
N
1)
N
N
O
OBn
OBn
2
) Ac O
3
steps 9d
Amberlyst
2
O
Cbz
11 Cbz
13 Cbz
N
then
5 steps
OH
N
H
3) SeO2
+
2) DAST
+
+
O
AllO
F
HO
F
Boc
OH
8
O
O
OAc
(N-Boc)-7
N
N
N
1
1
1
1
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0
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0
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0
1
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8
9
0
1
2
3
4
5
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7
8
9
0
OBn
OBn
10d
12
14
Cbz
Cbz
Cbz
b) Fleet
O
F
F
F
F
(R)
F
XtalFluor-M,
O
HO
O
(R)
O
9 steps
9%
Et N.3HF, DCM
2 steps
3
O
O
CO Me
2
N
N
N
2
84%
OMe 55%
OH
O
H
HO
Bn
17
Bn
16
15
6
c) Our previous work
HO
HO
OH
F
F
F
(R)
F
(S)
NfF, TBAT,
O
OH
4
steps
59%
O
DIPEA, THF
O
1 step
O
N
N
N
N
H
OBn
24%
OBn 98%
OH
Boc
19a
Boc
20a
Boc
18
(N-Boc)-4
Scheme 1. Precedence for the synthesis of 3,4-difluoroprolines
In this work, we describe in detail the synthesis of the yet unreported (3S,4S)-3,4-
difluoroproline 5, and a novel, more concise route for (3R,4R)-3,4-difluoroproline 6, both as
their N-Boc derivatives, and as their N-acetylated methyl esters 21 and 22 (Scheme 2).
Following our earlier communication, the development of the synthesis of N-Boc-4, including
further optimization efforts of the bis-deoxyfluorination step as well as a direct synthesis of
(N-Fmoc)-4, are described. The ring pucker analyses, prolyl bond cis/trans ratios and
isomerization kinetics of 21 and 22 are described and compared to those of unmodified proline
and the four known monofluorinated proline derivatives. Since 5/21 can be regarded as a

combination of the (4R)-FPro and (3R)-FPro, both known to be biased to the C exo pucker and
14
trans peptide bond configuration relative to proline, it was anticipated that 5/21 will display
a conformational bias in the same direction. Similarly, 6/22 was expected to have a larger

proportion of the C endo pucker and of the cis peptide bond configuration relative to proline,
as it is a combination of (4S)-FPro and (3S)-FPro.
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RESULTS AND DISCUSSION
Retrosynthetic Analysis
0
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0
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8
9
0
O
HO
OH
O
O
or
N
N
OBn
OBn
PG
PG
9a/b (syn)
0a/b (anti)
19a/c (3R,4S), and
diastereomers
1
HO
F
F
F
F
O
O
O
O
N
N
OBn
OBn
N
N
OMe
OBn
PG
PG
Ac
PG
25a-c
26a-c
2
2
1b (3S,4S)
2b (3R,4R)
20a/c (3S,4R)
23a (3S,4S)
O
F
O
O
O
OBn
2
4a (3R,4R)
N
N
OBn
a PG = Boc
b PG = Ac
c PG = Fmoc
Boc
Boc
29a
2
2
7a (3R)
8a (3S)
Scheme 2: Retrosynthetic Analysis.
Our retrosynthetic analysis of 3,4-difluoroprolines is outlined in Scheme 2. Functional group
interconversion to epoxides 9a/b and 10a/b, as in the Novartis work, appeared attractive, as it
would allow direct epoxide opening with fluoride followed by deoxyfluorination of the
resulting fluorohydrin. Alternatively, diol 19a/c provided an approach for 3,4-difluorination,
with excellent precedence available from the Marson group, who obtained trans-3,4-
difluoropyrrolidine from trans-3,4-dihydroxypyrrolidine via the corresponding triflates.38
While the epoxides and diols would be accessed from 3,4-dehydroderivative 25a-c, direct
functionalization of 25a-c such as vicinal difluorination or a halofluorination/fluoride halide
displacement could also lead to the desired 3,4-difluoroprolines. 3,4-Dehydroproline is a
commercially available (expensive) building block but can also be obtained by a well-described
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elimination process involving 26a-c starting from cheap (4R)-4-hydroxyproline. Finally, an
3
9
electrophilic fluorination approach as recently described by Ciulli et al. leading to 27a/28a
was also envisaged. With facile deprotection and versatility in mind, a benzyl ester in
combination with various amine protecting groups were used throughout our investigations.
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
,4-Dehydroproline Synthesis
X
O
DBU (3 equiv)
toluene, 110 ºC
O
O
+
N
N
N
OBn
OBn
OBn
Ac
Ac
Ac
MsCl,
pyridine
quant)
26b X = OH
0b X = OMs
(±)-25b (53%) (±)-31b (25%)
3
(
HO
O
o-NO -C H SeCN
O
2
6
4
O
N
N
N
PBu , THF;
OBn
3
OBn
OBn
PG
H2O2
PG
PG
25a (63%)
25c (78%)
2
26c
6a
a PG = Boc
c PG = Fmoc
31a
31c
Scheme 3. Synthesis of Protected 3,4-Dehydroproline 25a-c.
Initial efforts focused on achieving a large-scale synthesis of 3,4-dehydroproline 25. Following
a literature protocol, conversion of protected (4R)-4-hydroxyproline 26a to the corresponding
4
0
iodide, via a Mitsunobu reaction, followed by DBU-promoted HI-elimination, gave a ±5:1
mixture of alkene regioisomers from which the desired alkene 25a could be isolated in
excellent combined 76% yield (not shown), with 16% of the undesired 4,5-alkene 31a. While
this elimination reaction gave 25a as a pure enantiomer (>97% ee, see Supporting Information),
the separation of the alkene isomers was cumbersome. Moreover, it was found that conversion
of 26b to the corresponding 4-OMs derivative 30b (Scheme 3), followed by elimination using
the same base, led to a mixture (±2:1 ratio) of racemic alkene 25b and partially racemized 31b.
A 89:11 ratio of amide rotamers of 31b was observed in the NMR spectra, with NOESY
analysis showing the trans-isomer being the major rotamer (see Supporting Information).
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
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4
4
4
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5
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5
5
5
5
5
6
Pleasingly, a one-pot Grieco elimination sequence41 directly starting from 26a gave
enantiopure 25a as the major regioisomer with increased regioselectivity (>10:1 ratio), and
with negligible degree of racemization. The smaller amount of 31a facilitated purification
considerably. Furthermore, in contrast to base-mediated elimination reactions, it was found
that direct Grieco elimination of (4R)-4-hydroxyproline could also be performed with an N-
Fmoc protecting group (26c) in very good yield. This procedure is an improvement over the
previously reported 2-step elimination via 4-SePh intermediates, which are typically prepared
from the corresponding 4-OMs or 4-OTs derivatives.42-46
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Vicinal Difluorination and Halofluorination
Direct vicinal difluorination of alkene 25a was attempted using recent methods developed by
Gilmour and Jacobsen, both based on the in situ generation of a hypervalent iodoarene
difluoride.47-48 Unfortunately, both methods were unsuccessful and only led to recovered
starting material (not shown). Subsequent attempts to effect halofluorination on 25a using
different combinations of NBS, NCS or NIS with either HF•pyridine or Et N•HF were
3
unsuccessful as well, and this line of research was abandoned.
Epoxide-based Strategy
O
O
O
O
O
m-CPBA,
N
DCE
+
OBn
N
N
OBn
OBn
PG
PG
PG
2
2
5a
5b
a: PG = Boc
b: PG = Ac
10a (52%)
10b (55%)
9a (32%)
9b (25%)
Scheme 4. Synthesis of Protected 3,4-Epoxyprolines.
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Epoxidation of 3,4-dehydroproline derivatives is known, but not with the Boc/Bn or Ac/Bn
protecting group combinations. Following protocol, treatment of 25a/b with mCPBA led to a
mixture of epoxides 9a/b and 10a/b in good yield with the trans-isomer 10a/b isolated as the
major isomer after chromatography (Scheme 4). While determination of the epoxide
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
stereochemistry was achieved by H NMR analysis as reported by Robinson et al on N-Cbz-
4
9
3
,4-epoxyproline benzyl esters (Supporting Information), unambiguous conformation of the
stereochemistry was obtained by X-ray crystallographic analysis of 9b (Supporting
Information).
Table 1 Conditions investigated for the direct fluoride opening of epoxides 10a and 10b.
O
X
OH
HO
O
O
O
O
conditions
+
+
N
N
N
N
OBn
OBn
OBn
OR
PG
PG
PG
34a
34b
H
a: PG = Boc
b: PG = Ac
1
0a
32a X=Cl
33b X=F
35 R=Bn
10b
36 R=CH CH OH
2 2
Entry
PG Conditions
T (°C) Time (h) Product
1
2
3
4
5
6
7
8
Boc Et
Boc Et
Boc Et
Boc Et
3
3
3
3
N•3HF (neat)
130
72
35 (quant)b
N•3HF/THF (2:1), MWc 100
N•3HF/THF (2:1), MWc 100
0.08
0.33
0.66
10a (86%), 35 (7%)b
10a (72%) and 35 (28%)b
N•3HF/THF (3:1), MWc
35 (quant)b
130
Ac
Ac
DMPU•HF, DCM
DMPU•HF, DCE
rt50 43
10b (30%), 33b (15%)
10b (25%), 33b (5%)
32a (74%)
60
72
25
24
Boc Bu
Boc Bu
4
4
NH
NH
2
2
F
F
3
3
, DCE
120
120
, toluene
34a (56%)
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
Ac
TBAF, t-BuOH
70
4
34b (30%), 35 (13%)
36 (59%), 35 (2%)
10
Boc KHF
2
, ethylene glycol
150
22
a
Severe gel formation. b Calculated yields based on H NMR analysis of the crude reaction
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
mixture. Microwave irradiation.
First, epoxide 10a was investigated as a substrate for direct fluoride opening with HF-reagents
(
Table 1). Reaction with Et N•3HF in dichloroethane (DCE) at 80 °C for 3 days resulted in a
3
complex mixture of chlorinated and fluorinated products (±15%), alongside 68% of recovered
starting material (not shown), but conducting the reaction neat with increasing the reaction
temperature to 130 °C (Entry 1) induced deprotection and aromatization leading to pyrrole 35
in a quantitative yield. Due to its low reactivity, the use of Et N•3HF is often characterized by
3
long reaction times and high reaction temperatures, which can be alleviated by microwave
5
0
irradiation. However, with a short reaction time, no product was observed and increasing the
reaction time and temperature led to pyrrole 35 (Entries 2-4). With the more reactive
5
1
DMPU•HF, reaction of 10b did lead to fluorohydrin 33b in a 15% yield, together with 30%
recovered starting material (Entry 5). Unfortunately, raising the reaction time and temperature
did not improve the yield (Entry 6). These reactions suffered from gel formation which
impeded the isolation of the products. The use of hexafluoroisopropanol (HFIP) as an additive
successfully disrupted gel formation, but no fluorination was observed (not shown). Next,
epoxide opening was attempted with Bu NH F . Unexpectedly, reaction at reflux in DCE
4
2 3
yielded chlorohydrin 32a (Entry 7). Presumably, decomposition of the solvent under these
conditions must have released chloride ions, which subsequently opened the epoxide. In
toluene, Bu NH F was found to be too basic, with fluoride causing H -deprotonation leading
4
2
3
α
to the formation of allylic alcohol 34a (Entry 8). This was also the major pathway upon reaction
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with TBAF in t-BuOH (Entry 9). Interestingly, in contrast to the 4,5-dehydro isomer 31b, the
1
3
1
C- and H-NMR spectra of 34b only showed a single set of resonances, which could indicate
the presence of a single rotamer. The NOESY NMR spectrum of 34b is consistent with the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
trans-rotamer (Supporting Information). With KHF in ethylene glycol at 150 °C (Entry 10),
2
aromatization and transesterification was observed, yielding 36.
With direct fluoride opening being unsuccessful, it was then attempted to perform fluorination
after prior epoxide opening with different nucleophiles (Scheme 5). Precedence for opening of
5
2
proline epoxides includes reaction with MgI (78%) and 2-chloro-3-ethylbenzoxazolium
2
5
3
tetrafluoroborate (32%), both exclusively at the 4-position.
O
X
OH
HCl in dioxane (4M)
or
O
O
N
OBn
N
OBn
pTSA.H O, DCM
2
Ac
Ac
or
1
0b
32b X = Cl (95%)
37b X = OTs (73%)
MgBr or HBr, DCM
2
3
8b X = Br (88%, 95%)
X
OH
TsO
F
O
DAST,
DCM
O
O
+
N
OBn
N
OBn
N
OBn
Ac
Ac
Ac
3
3
2b X = Cl
7b X = OTs
39b
(40%, from 32b)
40b
(6%, from 37b)
(62%, from 37b)
X
OH
O
HO
O
TBAF,
O
O
OBn
+
tBuOH
N
N
N
OBn
OBn
Ac
Ac
Ac
3
3
7b X = OTs
8b X = Br
34b
10b
(48%, from 37b) (19%, from 37b)
(84%, from 38b)
AgF, CH NO (quant)
3
2
Scheme 5. Epoxide opening with other nucleophiles and subsequent fluorination attempts.
Starting from 10b, regioselective opening with HCl, HOTs, and HBr (or MgBr ) led to the
2
corresponding 4-substituted 3-hydroxyprolines 32b, 37b, and 38b in excellent yields.
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
However, subsequent DAST-mediated deoxyfluorination reactions mostly led to
aromatization: for the chlorohydrin 32b, pyrrole 39b was the only product isolated, while with
the β-hydroxy tosylate 37b, a low yield of the desired 3-fluorinated product 40b was obtained,
alongside 62% of pyrrole 39b. Tentative assignment of the expected stereochemistry of 40b at
C was based on the observed coupling constant of 5 Hz between H and H . Attempts to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
β
α
β
achieve fluorination at the 4-position in the presence of the 3-OH group by bromide or tosylate
displacement with TBAF-t-BuOH were also unsuccessful. Starting from 37b, a mixture of
allylic alcohol 34b and epoxide 10b was obtained. Despite the reduced basicity due to
hydrogen bonding with t-BuOH, fluoride must have deprotonated the alcohol group of 37b
causing epoxide formation, followed by H -deprotonation, resulting in epoxide opening to give
α
3
4b. Using bromohydrin 38b, the same allylic alcohol 34b was the only product isolated.
Interestingly, treating 38b with AgF in nitromethane only led to epoxide formation in
quantitative yield.
At this point, the epoxide-based strategy was abandoned, and attention shifted to fluorine
introduction via a vicinal diol group.
Direct Bis-deoxyfluorination Approach
Table 2. Dihydroxylation of 3,4-dehydroproline 25a/c.
HO
OH
HO
OH
O
See
O
O
N
+
OBn
N
N
Table
OBn
OBn
PG
PG
PG
2
2
2
5a
5c
5d
a PG = Boc
c PG = Fmoc
d PG = Cbz
19a
19c
9d
41a
41c
41d
1
Entry PG
Conditions
Yield 19a/c
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Boc
OsO , NMO, H O/dioxane (1:4)
92%
94%
82%
66%
80%
4
2
Boc K OsO 2H O, NMO, H O/acetone (1:3)
2
4•
2
2
Boc
Boc
AD-mix α, t-BuOH/H O (1:1)
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
AD-mix β, t-BuOH/H O (1:1)
2
Fmoc K OsO •2H O, NMO, H O/acetone (1:3)
2
4
2
2
Dihydroxylation of Cbz-protected 3,4-dehydroproline 25d with OsO has been reported to be
4
high-yielding and very stereoselective leading to the 2,3-trans-2,4-trans-diol 19d as the major
isomer.44-45, 54 A similar result was observed when these conditions were applied to 25a (Table
2
, Entry 1). Interestingly, starting from the Boc-protected 25a with the osmate ester (Entry 2),
3
4
no all-cis-diol 41a was observed. As both diastereomeric cis-diols were desired, attempts to
5
5
promote formation of all-cis-diol 41a using Sharpless asymmetric dihydroxylation conditions
were carried out. However, reacting 25a with both AD-mix- and AD-mix- only led to the
formation of 19a in 82% and 66% yield respectively (Entries 3-4). Finally, dihydroxylation
was also carried out on the Fmoc-protected alkene 25c using the osmate ester conditions, also
exclusively leading to N-Fmoc-protected 2,3-trans-2,4-trans-diol 19c (Entry 5).
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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Marson: HO
OH
TfO
OTf
F
F
Tf O, DCM
TBAF
2
N
N
N
Bn
Bn
Bn
4
2
43
44
HO
OH
TfO
OTf
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
Tf O, DCM
2
N
N
N
OBn
OBn (64%)
OBn
Boc
Boc
Boc
1
9a
45a
(not observed)
39a
HO
HO
OH
F
F
O
+
ONf
O
O
NFF, TBAT
N
N
N
OBn DIPEA, THF
OBn
OBn
Boc
46a (23%)
Boc
Boc
1
9a
20a (24%)
OH
O
O
O
NFF, Et N.3HF,
O
3
S
C4F9
F
N
N
OBn
OBn Et N, CH CN
3
3
H
NFF
Fmoc
9c
(26%)
3
5
1
Scheme 6. Fluorination of Dihydroxyproline via Sulfonate Intermediates.
Marson et al previously demonstrated that starting from a trans-3,4-ditriflate substituted
pyrrolidine ring 43 (Scheme 6), vicinal difluorination with TBAF can yield the corresponding
trans-3,4-difluoropyrrolidine 44 in good yield,38, 56 and this transformation has also been
5
7
successful on the corresponding Cbz derivative. However, treatment of 3,4-dihydroxyproline
1
9a with triflic anhydride already resulted in the formation of pyrrole 39a in a 64% yield.
Hence, reaction with nonafluorobutanesulfonyl fluoride (NfF)58 in combination with
tetrabutylammonium difluorotriphenylsilicate (TBAT)59 was attempted, as this process
generates sulfonates in the presence of fluoride. Pleasingly, this led to 20a as the only observed
1
9
3
,4-difluoroproline diastereoisomer ( F NMR analysis), with an enol sulfonate 46a as major
byproduct along with its hydrolysis product, 3-oxoproline, as a minor, but persistent, impurity
not shown). Interestingly, no pyrrole side product was observed. While separation of all
(
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products was possible by HPLC, purification was considerably facilitated by subjecting the
reaction mixture to NaBH in order to reduce the 3-oxoproline byproduct to the corresponding
4
alcohol (not shown). The regiochemistry of enol sulfonate 46a was established by means of a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
D HOESY NMR experiment.
As a Fmoc protecting group does not tolerate basic conditions, TBAT could not be used as a
fluoride source for the NfF fluorination. Even when (diluted) Et N•3HF/Et N was employed
3
3
6
0
19
as a fluoride source, no difluorination was observed in the crude F NMR and pyrrole 35
was the only product obtained from the reaction.
O
O
O
S
N
HO
OH
F
F
O
F OH
O
1
) DAST, DCM, 3d
O
O
O
O
+
+
+
OBn
N
OBn
2) NaIO , RuCl ,
N
4
3
N
N
OBn
OBn
OBn
CH CN/H O
PG
3
2
PG
PG
PG
PG
48a
48c
a PG = Boc
c PG = Fmoc
1
19c
9a
20a (26%)
20c (14%)
47a
47c
39a
39c
Scheme 7. Fluorination of Dihydroxyproline Using DAST.
The reaction of the 3,4-diols 19a and 19c was also investigated with DAST (Scheme 7). With
9a, this led to a complex reaction mixture in which the desired difluorinated 20a was clearly
1
visible by 19F NMR analysis, next to two minor byproducts, which presumably were
monofluorinated hydroxyfluoroprolines 47a. As the desired 20a co-eluted with another
byproduct, identified as the corresponding cyclic sulfite, the crude reaction mixture was
subjected to typical oxidation conditions leading to the formation of the cyclic sulfate 48a.
Isolation was now possible, leading to 20a in 26% yield. According to MS-analysis, the sulfite
oxidation was not accompanied by possible61 proline C5-oxidation to the corresponding
lactam. Similarly, when this sequence was applied to the Fmoc-protected 19c, the desired 3,4-
difluoroproline 20c was also isolated, albeit in a reduced 14% yield.
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Despite the low yield of this double deoxyfluorination process, the very short synthesis (only
three steps from protected (4R)-hydroxyproline) was deemed an acceptable and practical
synthesis, as gram-scale quantities of 20a could readily be obtained.
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Electrophilic Fluorination Strategy
With no straightforward access to other 3,4-dihydroxyproline diastereoisomers as substrates
for bis-deoxyfluorination, investigations turned towards an electrophilic fluorination approach.
Barraclough et al had demonstrated the regioselective conversion of a 4-ketoproline derivative
to the corresponding silyl enolether,62-63 which was used to stereoselectively introduce
deuterium at C3. Hence, formation of the silyl enol ether 49a was achieved upon treatment of
29a,64,65,66 synthesized by Dess-Martin periodinane oxidation of 26a in 94% yield (not shown),
with LDA and TMSCl, and subsequently fluorinated with SelectFluor (Scheme 8). In our
hands, this transformation proved to be low yielding and was found difficult to optimize,
leading to a mixture of isomers 27a/28a in maximum 31% yield. Reduction of the 4-keto group
led to a mixture of two separable fluorohydrin isomers, 50a and 51a, in moderate yield. In the
course of the optimization process, Ciulli and co-workers reported the synthesis of 27a/28a in
5
0% yield using this procedure, and of 50a/51a in 58% and 30% yield respectively.39
Interestingly, they also isolated a third diastereomer. Preliminary assignment of the
stereochemistry at C was based on the observed coupling constant between H and H , which
β
α
β
was 6 Hz for 50a and 2 Hz for 51a. This value for 50a is in line with the coupling constant
observed between H and H in 20a. In addition, for 50a, clear NOESY cross peaks where
α
β
observed between H and H and between H and H , suggesting all protons are on the same α-
α
β
β
γ
face of the pyrrolidine ring. This assignment was in agreement with the Ciulli work.39
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Scheme 8. Electrophilc fluorination route to 23a and 24a.
Deoxyfluorination of both 50a and 51a was achieved in very good yield by treatment with the
NfF and TBAT reagent combination. The stereochemistry of 24a was unambiguously assigned
by means of X-ray analysis (Figure 3).
Figure 3. X-ray structure of (3R,4R)-3,4-difluoroproline 24a. Thermal ellipsoids drawn at the
5
0% probability level.
With the new 3,4-difluoroproline derivatives 23a and 24a in hand, conversion to the required
N-acetyl methyl ester derivatives 21 and 22 was carried out to allow conformational studies,
including comparison with other, known, N-acetylated fluoroproline methyl esters.10, 13, 17, 67
Hence (Scheme 9), the benzyl protecting group was removed by hydrogenolysis, and the N-
Boc group by treatment with methanolic HCl. These conditions also simultaneously effected
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methyl ester formation. Finally, the amine groups were converted to their corresponding N-
acetyl derivatives 21 and 22.
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0
Scheme 9. Synthesis of N-acetyl methyl ester derivatives 21 and 22.
It was possible to obtain single crystals of 21, and crystallographic analysis (Figure 4) provided
unambiguous proof of its relative stereochemistry.
Figure 4. Crystal structure of (3S,4S)-3,4-difluoroproline 21. Thermal ellipsoids drawn at the
50% probability level.
Conformational and Kinetic Analyses
The experimental trans:cis ratios in chloroform and water, the experimental cis-trans
isomerization rate constants in water, and the DFT-calculated pucker preferences for the N-Ac-
X-OMe model compounds of proline, the (3S,4R)-, (3R,4R)- and (3S,4S)-3,4-difluorinated
prolines and their monofluorinated progenitors are reported in Table 3. The entries are
organized according to pucker preference. The data for the (3S,4R)-variant 56 has been
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reported and discussed previously, but are included in Table 3 for the sake of completeness.
In the following discussion, the term ‘bias’ assumes the conformational preference of the non-
fluorinated N-acetyl proline methyl ester as a reference.
1
1
1
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The amide trans:cis ratios in both chloroform and water of the 3,4-difluorinated proline 21 are
very similar to those of each of their monofluorinated progenitors 52 and 53. For 22, the ratios
are closer to those of (4S)-fluoroproline 54 than the (3S)-derivative 55.
The cis-trans isomerization rates (represented here by k = k
+ ktrans-cis) typically increase
cis-trans
ex
with increasing number of fluorine substitutions, mostly due to the electron withdrawing effect
1
1
of the fluorine atoms decreasing the double bond character of the amide bond. As expected,
both the (3S,4S)- and (3R,4R)-difluorinated variants, 21 and 22, indeed show higher
isomerization rates than their monofluorinated progenitors. Interestingly, the (3R)-variant 53
29
has a markedly higher isomerization rate than all other monofluorinated prolines, and even
exchanging faster than the (3R,4R)-difluorinated variant 22. This remarkable acceleration by
fluorination at the 3-position with this stereochemistry is retained when combined with
fluorination at the 4-position, resulting in even higher isomerization rates for the (3S,4S)-
variant 21. The isomerization rate for 21 is also much higher than that of the previously
described (3S,4R)- and (4,4)-difluorinated variants.34
Table 3. Experimental trans-cis ratio’s and amide isomerization rates, and calculated
pucker ratio’s
F
F
F
(S)
F
(S)
H/F
(R)
(R)
H/F
O
O
O
N
Ac

CO Me
2
N
N
N
F
F
(S)
OMe
OMe
OMe
(R)
O
Ac
Ac
Ac
C -exo
52
53
F
21
N
OMe
F
F
(R)
F
(R)
(
S)
(S)
H/F
CO2Me
Ac
H/F
O
O
O
56
N
N
N
N
OMe
OMe
OMe
Ac
Ac
Ac
Ac
54
55
22

C -endo
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

i
K
trans:cis (exp.)
C endo: C exo (DFT)
CHCl
trans
81:19 90:10
-1
(25 °C)
cis/trans kinetics, 35°C (s ) (exp.)
3
2
H O
d
CDCl
3
D
2
O
k
cis-trans
k
trans-cis
k
ex
cis
trans
66:34
56:44
15:85
7:93
cis
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Ac-Pro-OMe
3.85b
3.72
5.08
4.26b
4.32
4.19
1.64b
1.98
4.62b 0.031e
0.007e
0.038
82:18
90:10
44:56
17:83
9:91
5
5
5
2
5
6 (3S,4R)
3 (3R)
5.00
0.119±0.009f
0.025±0.002f
0.019±0.003f,g
0.010e
0.144±0.011 41:59 78:22
0.159±0.024 24:76 16:84
8.31c 0.141±0.021f,g
6.74b 0.064e
2 (4R)
0.074
11:89 28:72
1 (3S,4S)a
5 (3S)
7.23
0.210±0.005f
0.031±0.001f
0.009±0.001f,h
0.015e
0.242±0.006 19:81 11:89
20:80
97:3
4.31c 0.030±0.004f,h
2.49b 0.037e
0.038±0.005 98:2
0.052 97:3
0.090±0.013 97:3
98:2
99:1
99:1
99:1
54 (4S)
99:1 99.5:0.5
99:1 99:1
22 (3R,4R)a
2.79
0.065±0.009f
0.024±0.003f
aNote that CIP prioritization changes with introduction of the second fluorine atom, so that 21 must be compared with 52 and
b
5
3, and 22 with 54 and 55. In good agreement with reported ratios by Siebler et al.10 cIn good agreement with reported ratios
by Kim et al.68 d
k
ex is defined as kex = kcis-trans + ktrans-cis. eCalculated value based on Renner et al.13 fExperimental NMR value
obtained using similar procedure as Renner et al.13 gCorresponding values reported by Thomas et al. at 37 °C using an
alternative experimental procedure: 0.229 s and 0.028 s–1.29 hCorresponding values reported by Thomas et al. at 37 °C using
–
1
an alternative experimental procedure: 0.065 s and 0.016 s–1.29 iDFT values, using the M06 functional with cc-pVDZ basis
–1
set and CHCl or water implicit solvent models.
3


Finally, the calculated ratios between C endo and C exo puckers using DFT with chloroform
or water as implicit solvent are provided (Table 3). Unmodified proline has a higher preference


9
for the C endo than the C exo pucker. Both (4S)- and (3S)-fluoroprolines, 54 and 55, strongly


bias these pucker ratios to the C endo form, with negligible C exo pucker populations, both
2
1
in chloroform and water. As expected, the (3R,4R)-difluoroproline variant 22 is heavily



biased to the C endo pucker as well, with essentially the same C endo: C exo ratio as that of
its (3S)- and (4S)-progenitors. The (4R)- and (3R)-fluoroprolines, 52 and 53, are biased to the

C exo pucker relative to Pro, albeit to different degrees. Where the (4R)-variant 52 shows a
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
similar C exo-bias in both solvents and for both trans and cis forms, the cis rotamer of the

(3R)-variant 53 shows a high C exo bias in chloroform, but a low bias in water. The (3S,4S)-

difluorinated proline 21 shows a bias to C exo pucker in the same order of magnitude as its
1
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
progenitors. Interestingly, especially in the cis rotamer, the C exo pucker is highly populated
in both solvents, even higher than in its trans rotamer and than in its progenitors.
Experimental verification of these computational results can in principle occur via analysis of
vicinal scalar couplings. Unfortunately, 3JFF couplings are known not to be practically
6
9
exploitable to assess the dihedral angle, while quantitatively calculating the ring pucker from
experimental 3JHF and 3JHH couplings was in our hands found not to be reliable due to the
limited accuracy of Karplus relations for difluorinated five-membered pyrrolidine rings.
Instead, these couplings can qualitatively be compared to those of the monofluorinated
progenitors (Table 4), bearing in mind that the different fluorine substitution patterns may
significantly influence the Karplus relation. The (4R)- and (4S)-monofluoroprolines, which are


established as strongly biased to respectively C exo and C endo, clearly display distinct
transoid 3JHH coupling constants of 8.2 /10.1
Hz and <1.0 Hz respectively, implying this
cis
trans
coupling provides a sensitive measure for the endo:exo ratio. Both the similar small magnitude

21
of this coupling in (3S)-monofluoroproline, known to have a pronounced C endo pucker,
and the larger values found for proline (2.6 /4.7 Hz), consistent with intermediate endo:exo
cis
trans
ratios and a higher endo population in the cis-form, confirm the relevance of 3JHH coupling
constants for a qualitative analysis of fluorinated proline ring pucker. Hence, given the (3R,4R)-
difluorinated variant 22 also shows a small 3JHH coupling value of <0.5 Hz, its calculated

preference for a C endo pucker is consistent with these experimental data.
In contrast, the cisoid 3JHH coupling constants of the (4R)- and (4S)-fluoroprolines and
proline show similar values of 8.7 /7.8
Hz, 9.7 Hz and 8.9 /8.8
Hz respectively,
cis
trans
cis
trans
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implying this coupling is not very sensitive to the endo:exo ratio. Indeed, both the (3R)-
2
1
fluoroproline, known to prefer an exo pucker, and the (3S,4S)-difluoroproline show lower
cisoid 3JHH couplings of 5.1/4.8 Hz and 5.2/5.0 Hz respectively, which suggests the fluorine
substitution pattern is in this case the most significant factor determining the value.
Nevertheless, the similarity of both the 3JHH and 3JHF couplings observed for the (3R)- and
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0
(3S,4S)-variants suggests both fluoroprolines have mostly similar endo:exo ratios. In addition,
these couplings differ significantly with those of the (3S,4R)-variant, which is expected given
the latter displays virtually no pucker preference.
Table 4. Comparison of the relevant coupling constants
3JHF (Hz)a
3JHH (Hz)a
cis-amide
trans-amide
cis-amide
trans-amide
Biasb
Transoid
transo cisoi transo cisoi transo
Cisoidc
cisoid
Compound
c
id
d
id
d
id
4.7
n/a
n/a
10.1
n/a
Ac-Pro-OMe
-
n/a
n/a
n/a
7.4
n/a
n/a
n/a
8.9
7.9
5.1
8.7
5.2
n/a
9.7
n/a
2.6
8.8
7.3
4.8
7.8
5.0
(
(
(
(
(
(
(
3S,4R) 56
-
13.7
28.2
n/a
n/a
3R) 53
exo
exo
exo
endo
endo
endo
n/a
25.9
n/a
n/a
n/a
4R) 52
n/a
n/a
8.2
3S,4S) 21
3S) 55
n/a
27.3
n/a
n/a
29.8
n/a
19.8
n/a
13.7
n/a
n/a
n/a
n/a
19
<1.0c
<1.0c
<1.0c
n/a
1.0
d
d
4S) 54
n/a
3R,4R) 22
21.2
n/a
24.4
n/a
<1.0c
a 1
1
70 1
H- H couplings measured using PSYCHEDELIC , H- F couplings were read from the 1D
1

b
H spectrum on the H proton. Bias refers to the conformational preference of the non-
c
fluorinated N-acetyl proline methyl ester as reference. “Cisoid” indicates that the coupled
c
atoms are on the same side of the proline ring, “transoid” on different sides of the ring. Value
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d

smaller than signal line width. Degenerate H chemical shifts, individual couplings could not
be extracted.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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1
2
3
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5
6
7
8
9
0

The clear C exo pucker bias observed for the (3S,4S)-difluorinated proline 21 in solution by
NMR is also observed in its crystal structure (Figure 4). A single crystal of 22 was not obtained,

but the C endo pucker bias of the (3R,4R)-difluoroproline ring could be observed in the crystal
structure of its N-Boc-protected precursor 24a (Figure 3). It should be noted that the packing
of molecules in the solid state, and their resulting conformations, is determined from the sum
of a multitude of inter- and intramolecular interactions, and often deviates from the
conformation in solution, which in turn is typically solvent dependent. With this caveat in
mind, the observed conformations in the crystal structures strongly suggest that the 3,4-
difluorination instills the expected conformational bias.
Discussion
The potential of fluorinated prolines as tools for protein research has a long track record. Next
to the well-known example of collagen, stabilized forms of proteins such as barstar,13
7
1
72
73
ubiquitin, Trp cage mini protein and GFP incorporating 4-fluoroprolines were obtained
with the C -stereochemistry selected to reinforce the pucker observed in the native protein.
4
Both 3- and 4-monofluorinated prolines have been used to probe the effect of -turn stability
6
8
on the self-assembly of elastin peptide mimics. Accelerated peptide folding, as a consequence
of the accelerated cis/trans kinetics, was observed when fluoroprolines were integrated in
thioredoxin (Trx),74 β2-microglobulin (β2m)75 and ribonuclease (RNase) A.76 Fluorinated
prolines have also been used to reveal the relevance of proline ring pucker in ribosomal peptide
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3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
synthesis.77-78 The extended range of cis/trans isomerization kinetics offered by the 3,4-


difluoroprolines — in conjunction with either a bias to trans and C exo pucker, to cis and C
endo pucker, or with similar structural preference to proline — clearly will be of interest within
such studies, allowing to deconvolute the roles of ring pucker and cis/trans preferences from
isomerization kinetics.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Recently, Bernardes and Corzana and coworkers used a rational Pro-to-FPro substitution to
7
9
stabilize an antigen-antibody complex. As a result of its proximity to a highly electronegative
fluorine, the polarization of a nearby CH-bond was increased. This led to an enhanced CH-π
interaction, which stabilized the antigen-antibody complex. A similar improved CH-π
interaction has been observed between a fluoroproline-modified phosphopeptide and the WW
domain of Pin1.80 Clearly, 3,4-difluorinated proline analogues, especially with 3,4-cis
stereochemistry, will be of great interest in that regard, as enhanced C-H polarization and thus
enhanced CH-π interactions can be expected.81
1
9
Regarding the use of fluoroprolines as F NMR reporters, the simultaneous fluorination at the
3
- and 4-positions provides for very distinct chemical shifts compared to the monofluorinated
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9
n
progenitors. The experimental F chemical shifts and J coupling constants for the N-Ac-X-
FF
OMe model compounds the (4,4)-difluoroproline, (3S,4R), (3R,4R)- and (3S,4S)-3,4-
difluorinated prolines, and their monofluorinated progenitors are shown in Table 5. For all 3,4-
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9
difluoroprolines, the homonuclear coupling constant between the vicinal F nuclei is small, as
opposed to that of the geminal difluorinated (4,4)-variant. This property is very useful for
advanced 19F NMR experiments, as it minimizes any potential complications from J-
modulation during spin-echo pulse sequences, or from second order effects, which is an issue
3
0
in geminal difluorinated prolines. In addition, the 3,4-difluorinated derivatives have very
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9
distinct F chemical shift values compared to their monofluorinated progenitors, even though
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they possess similar structural properties. The 3,4-difluoroprolines can thus be used
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9
complementary to the monofluoroprolines for F NMR purposes, allowing the design of
combinatorial incorporation schemes aimed at studying poly-proline and proline rich
sequences, due to maximum chemical shift dispersion between these residues, but with
minimal complications from homonuclear couplings.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Fluorine chemical shift values of fluorinated N-Ac-X-OMe derivatives (D O)
2
19F  /ppm (F3, F4)
nJFF (Hz)
Compound
Cis-amide
Trans-amide
-186.4
Cis-amide Trans-amide
(
3R) 53
3S) 55
-184.4
-176.8
-177.9
-173.1
-
-
-
-
(
-175.7
(4R) 52
-177.0
-
-
(
(
(
4S) 54
-172.9
-
-
3S,4S) 21 -195.0, -194.3 -197.2, -193.3
3R,4R) 22 -190.6, -187.7 -189.1, -188.4
11.1
12.9
5.8
236.0
11.4
13.0
4.7
233.7
(3S,4R) 56 -208.5, -200.3 -210.4 , -203.3
-101.4, -96.7a -98.2, -99.1a
(4,4) 57
a
pro-R F4, pro-S F4.
CONCLUSION
As part of a program to expand the scope of available fluorinated prolines, we report here in
full the effective syntheses of three 3,4-difluorinated proline analogues (as summarized in
Scheme 10). In addition, we report the first conformational characterization (trans:cis ratios
and isomerization kinetics, and ring-pucker preferences) of the (3R,4R)- and (3S,4S)-3,4-
difluoroproline analogues.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
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5
5
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6
The (3S,4R)-difluorinated proline derivative could not be synthesized directly from 3,4-
dehydroproline, or from the 3,4-epoxyproline derivative, with the former being unreactive
under conditions of alkene difluorination or halofluorination, and the latter typically suffering
from aromatization leading to pyrrole derivatives. However, a direct bis-deoxyfluorination
strategy with the easily accessible 3,4-dihydroxyproline as substrate led to the desired target
using both NfF and DAST, with the former giving the highest yield when N-Boc was used as
protecting group, and the latter suitable with an N-Fmoc protecting group. Yields were low
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(26% and 14% respectively), but as only two transformations were required from the protected
3
,4-dehydroproline, gram quantities are easily available. In this context, we report that the
direct synthesis of Fmoc-protected 3,4-dehydroproline from the corresponding 4-
hydroxyproline is possible using the one-pot Grieco elimination procedure, in contrast to the
usually employed basic conditions.
HO
HO
OH
F
F
DAST;
O
O
O
NaIO , RuCl
4
3
1
) Grieco-elimination
) Dihydroxylation
N
N
N
OBn
OBn
or
OBn
2
PG
PG
PG
NfF, TBAT
2
2
6a
6c
20a (24-26%)
20c (14%)
19a (59%, 2 st)
9c (62%, 2 st)
a PG = Boc
c PG = Fmoc
1
HO
F
F
F
NFF,
DMP
O
O
1
) LDA,
TMSCl
78 C
O
O
F
TBAT,
N
N
O
O
OBn
OBn
*
NaBH4,
-
Boc
Boc
N
N
OBn
OBn
50a (40%)
23a (78%)
2) Selectfluor
Boc
Boc
27a/28a
31% over
steps)
+
HO
F
F
F
29a (94%)
NFF,
O
O
(
2
TBAT,
N
N
OBn
OBn
Boc
Boc
51a (13%)
24a (72%)
Scheme 10. Summary scheme for the synthesis of protected 3,4-difluoroprolines.
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The (3R,4R)- and the novel (3S,4S)-difluorinated proline derivatives were synthesized using a
two-step fluorination strategy, the first being an electrophilic fluorination starting from
protected 4-ketoproline, and the second by a DAST-mediated deoxyfluorination after 4-
ketoreduction. Hence, starting from 26a, the (3S,4S)- and (3R,4R)-N-Boc 3,4-difluoroproline
benzyl esters were obtained in an overall yield of 9% for 23a and 3% for 24a, in a combined 5
steps (with three common steps before diastereomer separation). It may be pointed out that the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
linear 10-step Fleet synthesis of the (3R,4R)-N-Bn difluoroproline methyl ester 17 (cf Scheme
) has a higher overall yield (24%), although this delivers a single diastereomer only. In
1
addition, Ciulli reported higher yields for the electrophilic fluorination step.39 X-ray
crystallographic analysis allowed unambiguous determination of the relative configuration of
the obtained 3,4-difluoroprolines.
Due to the opposing conformational effects of each individual fluorine in the (3S,4R)-
difluorinated proline derivative, this analogue has previously been described as having minimal
3
4
conformational bias to proline. In contrast, it is shown here that a combination of 3- and 4-
fluorine substitutions with similar preorganizing effects, results in 3,4-difluorinated proline
derivatives with similar conformational preferences as monofluorinated prolines. While the
(3R,4R)-difluorinated proline derivative resembles most closely the (4S)-fluoroproline, the
(3S,4S)-difluorinated proline derivative resembles the (4R)-fluoroproline, though with a

19
somewhat higher preference for a C exo pucker in its cis rotamer. Given the distinct F
chemical shifts of both 3,4-difluorinated derivatives to their monofluorinated progenitors, they
will be of interest for multi-residue fluorine labelling strategies, for instance in the study of
repetitive or low-complexity protein sequences, where similar conformational preorganizing
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9
effects are desired, but distinct residue-specific F NMR chemical shifts are needed.
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