One-Pot Ring-Opening Peptide Synthesis Using α,α-Difluoro-β-Lactams

Article abstract of DOI:10.1055/s-0040-1707238

α,α-Difluoro-β-lactams successfully underwent ring-opening aminolysis with various amino acids in 2,2,2-trifluoroethanol to afford fluorine-containing peptides. In this aminolysis, it was found that 2,2,2-trifluoroethanol first attacked the α,α-difluoro-β

Full text of DOI:10.1055/s-0040-1707238

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2020, 52, A–J
paper
A
en
A. Tarui et al.
Paper
Synthesis
One-Pot Ring-Opening Peptide Synthesis Using
,-Difluoro--Lactams
Atsushi Tarui
DIPEA
(3 equiv)
Ar
Ar
O
NH
F
O
N
Masakazu Ueo
Marino Morikawa
Masahiko Tsuta
Sumika Iwasaki
Noriko Morishita
Yukiko Karuo
+
amino acid HCl salt
(2 equiv)
CF3CH2OH
(TFE)
F
Ph
AA OMe
Ph
F
F
fluorinated peptides
21 examples
• One-pot operation
• Properties of TFE:
up to 83% yield
1) Good leaving group via trifluoroethyl ester
2) Suitable acidity for the condensation
Kazuyuki Sato
Kentaro Kawai
Masaaki Omote*
Faculty of Pharmaceutical Sciences, Setsunan University,
Hirakata, Osaka 573-0101, Japan
omote@pharm.setsunan.ac.jp
Received: 17.06.2020
Accepted after revision: 09.07.2020
Published online: 17.08.2020
a)
NH2
O
NH2
O
OH
DOI: 10.1055/s-0040-1707238; Art ID: ss-2020-f0329-op
NHOH
N
N
H
H
OH
bestatin
IC50 (APN) = 2.55 μM
O
OH
O
Abstract ,-Difluoro--lactams successfully underwent ring-opening
aminolysis with various amino acids in 2,2,2-trifluoroethanol to afford
fluorine-containing peptides. In this aminolysis, it was found that 2,2,2-
trifluoroethanol first attacked the ,-difluoro--lactams with cleavage
of lactam ring to form the corresponding open-chain 2,2,2-trifluoroeth-
yl esters as reactive intermediates. The trifluoroethyl esters were more
electrophilic compared with the corresponding methyl ester and there-
by accelerated the aminolysis with various amino acids to form -amino
acid peptides with ,-difluoromethylene unit.
aminopeptidase inhibitor 1
IC50 (APN) = 1.26 μM
OH
O
AcO
O
NH
O
O
OAc
O
OBz
O
HO
OH
paclitaxel
b)
Key words fluorine, peptide, -amino acid, ,-difluoro--lactam,
2,2,2-trifluoroethanol
NH2
F
O
R
Ar
N
OH
N
+
amino acid
H
F
F
-Amino acids are considered important non-proteino-
genic amino acids and are deliberately incorporated into
peptides to change their physical properties and biological
activity.1 In drug discovery, -amino acids have been used
to mimic natural peptide-based antibiotics that have high
proteolytic stability in vivo.2 Protease resistance is an in-
trinsic property of -amino acids and has been successfully
exploited to produce the aminopeptidase inhibitor, besta-
tin3 and the more potent analogue 14 (Figure 1). We have
previously focused on these -amino acid structures, par-
ticularly 3-amino-2-hydroxy-3-phenylpropanoic acid moi-
ety, which is found in paclitaxel (Taxol®)5 and docetaxel
O
Ph
F
3
2
Figure 1 a) The structures of bestatin and its more potent analogue 1
and paclitaxel. b) Target compounds 3 and the retrosynthetic strategy.
Regarding the synthesis of -amino acid-containing
peptides, recent progress in ribosome or enzyme engineer-
ing allows -amino acid incorporation into polypeptides.8,9
However, in most cases, organic synthesis is still required to
prepare highly functionalized -amino acids and incorpo-
rate them into peptides. To date, there have been several re-
ports on the synthesis of functionalized -amino acids and
related structures,10 including those with fluorine substitu-
ents adjacent to the carbonyl (2-)11 or to the amino (3-)12
positions. -Lactam synthon method has been also known
for the construction of -amino acid substruc-
ture.10a,11a,c,e,f,13 However, to the best of our knowledge, there
are only few examples on the one-pot synthesis of
(Taxotere®).6 Additionally, we envisioned that
a hy-
droxymethylene group could be replaced by a difluoro-
methylene unit, which may be useful because fluoroalkyl
group sometimes behaves like hydroxyl groups in biological
assays.7 However, a convenient approach to synthesize pep-
tide 3 is still very challenging using current peptide chemis-
try.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
A. Tarui et al.
Paper
Synthesis
functionalized -amino acid-containing peptides, except
for conventional peptide synthesis, which involves sequen-
tial, step-by-step condensation reactions of activated amino
acid derivatives.11c,13c In this context, we hypothesized that
,-difluoro--lactams 2 ought to be a good precursor for
the one-pot synthesis of 3 via a ring-opening nucleophilic
attack by various amino acids, although the product would
be a diastereomixture.
For this approach, the synthetic drawback remained in
the reactivity of both 2 and the amino acid. Particularly, the
nucleophilicity of the nitrogen atom in the amino acid is
significantly lower than commonly used amino com-
pounds, so the direct reaction of 2 and amino acid would be
suppressed. To check the reactivity of 2 towards amino ac-
ids, 2a was reacted with glycine methyl ester hydrochloride
in the presence of N,N-diisopropylethylamine (DIPEA) as a
non-nucleophilic base (Table 1). The desired substrate 2
was synthesized by our previously reported Rh-catalyzed
imino-Reformatsky reaction of ethyl bromodifluoroacetate
using diethylzinc in a gram scale reaction.14
ever, the reaction rate was still slow (20 h). A dramatic im-
provement in reaction efficiency was observed when it was
performed in 2,2,2-trifluoroethanol (TFE) solution; 3a was
provided in 84% yield after two hours reaction time. How-
ever, when equimolar amount of TFE was used in methanol,
a significant decrease in yield of 3a was observed along
with unreacted 2a (entry 7). This reaction was very simple
to perform, and there was no need for any activation pro-
cess prior to the reaction, suggesting that this new ap-
proach was practical and effective for preparing ,-difluo-
rinated -amino acid containing peptides.
With the optimized conditions in hand, the substrate
scope of the reaction was examined. The results are shown
in Figure 2. All the reactions were completed within 2–3
hours. It is noteworthy that this reaction could accommo-
date -amino acids with bulky substituents. Not only sim-
ple amino acids, such as alanine, but also bulkier amino ac-
ids, valine, leucine, and isoleucine, all successfully reacted
with 2a to give peptides 3c–e in good yields. Hydroxy
groups on aliphatic carbons disturbed the reaction to some
extent, and their yield decreased to around 35% (→ 3f and
3g). However, the less nucleophilic phenolic group did not
disturb the reaction process, and 3h was obtained in good
yield. Other aromatic amino acids and basic amino acids,
which were masked amino nucleophiles on the side chain,
were also compatible (→ 3i–l). It is worth mentioning that
histidine was able to participate in the reaction without any
protection on nitrogen atom to give 3m in good yield. Ester,
amido, and sulfide substituents, and even nucleophilic thiol
in the case of cysteine, were compatible with the reaction
(→ 3n–s).
Table 1 Synthesis of ,-Difluoro--lactam 2a and Solvent Screen for
Ring-Opening Peptide Synthesis Using 2a as an ,-Difluoro--amino
Acid Unita
RhCl(PPh3)3 (1 mol%)
Et2Zn (3 equiv)
Ph
Ph
O
Ph
N
N
BrCF2COOEt
(2 equiv)
+
THF
F
Ph
F
2a (64%; 1.6 g after
recrystallization)
DIPEA
(3 equiv)
Ph
Ph
Ph
O
NH
O
N
+
H-Gly-OMe·HCl
(2 equiv)
N
CO2Me
F
solvent
Ph
H
Unfortunately, in the case of proline, the reaction was
unfavorable, and only a small amount of the product 3t was
observed as the reaction progressed; furthermore, it did
not appear to be since it decomposed in the purification
process. In the case of another bulky amino acid, phenyl
glycine, the reaction proceeded but longer reaction time
was needed to obtain 3u, which is an analogue of amino-
peptidase inhibitor 1, in 81% yield. It is interesting to note
that the tripeptide, H-Gly-Gly-Gly-OMe was able to react
with 2a to give 3v in good yield, highlighting the good per-
formance of this reaction as a convenient method for one-
pot peptide synthesis.
F
F
F
2a
3a
Entry
Solvent
Time (h)
Yield (%)b3a Recovery (%) 2a
1
2
3
4
5
6
7c
DMA
20
24
21
48
20
2
45
61
75
88
92
84
49
52
36
23
6
NMP
DMSO
DMF
MeOH
CF3CH2OH
MeOH
0
0
4
50
To demonstrate the high practical potential of this
method, we proceeded to synthesize the tripeptide 6 that
contained ,-difluoro--amino acid unit between two gly-
cine terminals. As shown in Scheme 1, N-PMP-,-difluoro-
-lactam (2b) was used instead of 2a to obtain a free nitro-
gen atom for consequent peptide elongation. As expected,
the reaction of 2b with H-Gly-OMe occurred in the same
manner, and peptide 4 was obtained. The crude product 4
was treated with ceric ammonium nitrate to remove the
PMP group and to give 5 in good yield (from 2b).15 For the
following peptide elongation at the N-terminus of 5, Fmoc-
Gly-OH was condensed with 1-hydroxybenzotriazole
a Reaction was done with the concentration of 0.67 M (0.2 mmol/0.3 mL) of
2a.
b Isolated yields.
c TFE and 2a were used in equimolar amounts.
The ring-opening aminolysis with glycine proceeded to
give the product of the open-chain peptide 3a. In this reac-
tion, polar aprotic solvents commonly used in SN2-type re-
actions seemed to decrease the reactivity and gave 3a with
very slow reaction times, which were impractical for use
(Table 1, entries 1–4). In contrast, methanol, a protic sol-
vent, accelerated the reaction to give 3a in 92% yield; how-
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

C
A. Tarui et al.
Paper
Synthesis
PMP
Ph
Ph
Ph
Ph
Ph
DIPEA
(3 equiv)
PMP
Ph
O
NH
F
O
NH
F
O
CH3
NH
F
O
NH
F
O
N
H-Gly-OMe·HCl
(2 equiv)
+
OMe
N
F
TFE
Ph
N
CO2Me Ph
N
H
CO2Me
N
CO2Me
H
F
F
O
H
H
F
F
F
2b
4
3b
3c
3d
(2 h, 77%)
(2 h, 72%)
(2 h, 70%)
CAN
(3 equiv)
Fmoc-Gly-OH (1.05 equiv)
HOBt, EDC
NH2
F
O
OMe
H
O
Ph
Ph
H
O
Ph
Ph
Ph
Ph
N
OH
OH
MeCN/H2O
H
DMF
NH
F
NH
F
O
N
NH
F
F
O
5 (70% from 2b)
N
CO2Me
Ph
CO2Me
N
CO2Me
H
F
H
F
H
F
O
H
F
F
H
N
3e
3f
3g
(2 h, 45%)
Fmoc
N
N
(2 h, 81%)
(2 h, 35%)
OMe
H
OH
H
N
O
Ph
O
6 (80%)
Ph
Ph
Ph
Ph
NH
O
NH
F
O
N
NH
O
Scheme 1 Tripeptide synthesis using 2b as the -amino acid synthon
with an ,-difluoro unit
Ph
N
H
CO2Me
CO2Me Ph
N
CO2Me
H
H
F
F
F
F
F
3h
(2 h, 83%)
3i
(3 h, 74%)
O2N HN
3j
(3 h, 73%)
NH
NH
(Scheme 2). When 2a was dissolved in TFE or methanol, re-
spectively, the ring-opening alcoholysis of 2a occurred at
almost the same rate in both cases. After confirming that
both conversions to TFE ester 7 or methyl ester 8 were com-
pleted in two hours (monitored by TLC), H-Gly-OMe and
DIPEA were added to each solution to take place the subse-
quent aminolysis.17 Interestingly, large differences were ob-
served in the reaction rate; the aminolysis in TFE solution
was obviously faster than that in methanol solution, and
gave 3a in 72% yield (compared to 26% in methanol solu-
tion). The acceleration effect could be attributed to the TFE
group being a much better leaving group for aminolysis, be-
cause of the existence of a strong electronegative trifluoro-
methyl group; pKa (TFE) = 12 versus pKa (MeOH) = 16.18 In
addition, the moderate acidity of TFE could work as an acid-
ic catalyst for aminolysis process. We consequently ascribed
the rate acceleration in TFE solution to the synergistic ef-
fects of an enhanced leaving group and acid catalysis, both
resulting from TFE.
NHBoc
H
N
Ph
Ph
Ph
Ph
Ph
N
NH
F
O
NH
F
O
NH
F
O
N
N
H
CO2Me Ph
N
H
CO2Me
CO2Me
H
F
F
F
3k
3l
3m
(3 h, 82%a)
(3 h, 80%b)
(3 h, 73%)
CO2tBu
Ph
Ph
Ph
Ph
CO2tBu
CONH2
CO2tBu
NH
O
N
NH
F
O
N
NH
O
Ph
CO2tBu Ph
N
H
CO2Me
H
H
F
F
F
F
F
3n
3o
3p
(2 h, 76%)
(3 h, 57%)
(2 h, 31%)
CONH2
Ph
SMe
Ph
Ph
Ph
Ph
SH
NH
F
O
N
NH
F
O
N
NH
F
O
N
CO2tBu
Ph
CO2Me
CO2Me
H
F
H
F
H
F
3q
3s
3r
(2 h, 76%)
(3 h, 71%)
(2 h, 74%)
Ph
Ph
Ph
NH
O
NH
O
CO2Me
NH
F
O
N
Ph
N
Ph
Gly Gly Gly OMe
Ph
CO2Me
F
F
F
F
H
F
3u
3v
3t
(6 h, –c)
DIPEA
H-Gly-OMe
TFE
(24 h, 81%)
(2 h, 82%)
DIPEA
TFE
Ph
Ph
NH
F
O
Figure 2 Scope of the reaction of 2a with various -amino acids esters.
Isolated yields are shown. a Amino acid ester (1 equiv) and DIPEA (2
equiv) were used. b Histidine methyl ester hydrochloride 2 (2 equiv) and
DIPEA (5 equiv) were used. c A complex mixture was obtained, but a
trace amount of 3t was detected in 1H and 19F NMR spectra.
2 h
0.5 h
O
CF3
F
7
Ph
almost quantitative conversiona
Ph
Ph
O
NH
O
N
Ph
N
H
CO2Me
F
F
F
F
via 7: 72%
via 8: 26%
2a
3a
DIPEA
H-Gly-OMe
MeOH
DIPEA
MeOH
Ph
(HOBt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiim-
ide (EDC) to give 6 in good yield.11d,16 This reaction scheme
exhibits the potential utility of our method by showing its
capability of preparing polypeptides containing an ,-di-
fluoro--amino acid unit at the desired position.
NH
F
O
2 h
0.5 h
Ph
OMe
F
8
almost quantitative conversiona
To elucidate the reaction mechanism, we tried to under-
stand how TFE accelerated the reaction between 2a and the
-amino acids. For this purpose, a kinetic study of the reac-
tion was performed in both the TFE and methanol solution
Scheme 2 Mechanistic study based on the reaction rates between TFE
and methanol solutions. a The conversion was confirmed by TLC analy-
sis, in which 2a had disappeared completely and any other spot arising
from 2a was not observed except for the formation of 7 or 8.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

D
A. Tarui et al.
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Synthesis
In conclusion, we have developed a convenient method
for the synthesis of -amino acid peptides containing an
,-difluoromethylene unit by using ,-difluoro--lact-
ams 2a and 2b. This ring-opening reaction allows access to
a wide range of fluorinated -amino acid peptides. Most -
amino acids, except for proline, were successfully used in
this transformation to provide the fluorinated dipeptides in
good yields. Additionally, the reaction mechanism was ex-
amined by comparing the reactivity in TFE and methanol
solutions, which demonstrated the activation process in
TFE. Further studies, for example, on the synthesis of par-
ticular peptide-based pharmaceuticals and analogues in
diastereomerically pure form, are ongoing in our laboratory
with enantiomerically pure 2.
2 h. The mixture was poured into H2O and then extracted with EtOAc.
The combined organic phases were washed with brine, dried
(MgSO4), and concentrated in vacuo. The crude mixture was purified
with flash column chromatography to give the pure product 3.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
glycinate (3a)
Eluent: EtOAc/hexane (3:7); yield: 58.5 mg (84%); colorless solid; mp
153.0–154.0 °C; Rf = 0.2 (EtOAc/hexane, 3:7).
1H NMR (DMSO-d6, 400 MHz):  = 9.07 (m, 1 H), 7.52 (d, J = 7.2 Hz, 2
H), 7.36–7.29 (m, 3 H), 7.02 (t, J = 8.0 Hz, 2 H), 6.73 (d, J = 7.6 Hz, 2 H),
6.55 (t, J = 7.4 Hz, 1 H), 6.27 (d, J = 10.4 Hz, 1 H), 5.28 (ddd, J = 19.5,
10.4, 9.4 Hz, 1 H), 3.88 (m, 2 H), 3.58 (s, 3 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 169.1, 163.3 (t, J = 28.8 Hz),
146.6, 135.2, 128.7, 128.6, 128.0, 117.2, 115.9 (dd, J = 260.3, 254.3
Hz), 113.7, 58.0 (dd, J = 27.5, 21.7 Hz), 51.7, 40.7.
19F NMR (DMSO-d6, 376 MHz):  = –107.0 (dd, J = 252.9, 9.4 Hz, 1 F),
–117.3 (dd, J = 252.9, 19.5 Hz, 1 F).
MS (EI): m/z = 348 [M]+.
HRMS (EI): m/z [M]+ calcd for C18H18F2N2O3: 348.1285; found:
348.1288.
NMR spectra were obtained from a solution in DMSO-d6 or CDCl3 us-
ing 400 MHz for 1H NMR, 100 MHz for 13C, 376 MHz for 19F NMR.
DMSO-d6 solution of NMR samples were recorded at 40 °C, unless
noted otherwise. Chemical shifts of 1H and 13C NMR are reported in
ppm downfield of TMS (1H = 0.00) and DMSO-d6 (1H  = 2.49, 13C  =
39.5). Chemical shifts of 19F NMR are reported in ppm from CFCl3 as
an internal standard. 13C NMR spectra were obtained with 1H decou-
pling. All data are reported as follows: chemical shifts, multiplicity
(standard abbreviations), coupling constants (Hz), and relative inte-
gration value. The measurement of all NMR spectra were obtained
from the diastereomixture of peptide products. HRMS experiments
were measured on a double-focusing mass spectrometer with an ion-
ization mode of EI or positive-FAB using glycerin as a matrix. All ex-
periments were carried out under an argon atmosphere in flame-
dried glassware using standard inert techniques for introducing re-
agents and solvents unless otherwise noted. All commercially avail-
able materials were used as received without further purification.
Solvents were heated to reflux over CaH2 (DMF) and Mg metal (TFE
and MeOH) under an argon atmosphere and collected by distillation
just before use.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-alaninate (3b)
Eluent: EtOAc/hexane (3:7); yield: 56.1 mg (77%); dr 1:1.3; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.00 (t, J = 6.0 Hz, 1 H), 7.53–7.50
(m, 2 H), 7.36–7.26 (m, 3 H), 7.04–7.00 (m, 2 H), 6.75–6.71 (m, 2 H),
6.57–6.53 (m, 1 H), 6.30–6.27 (m, 1 H), 5.34–5.22 (m, 1 H), 4.37–4.28
(m, 1 H), 3.57 (s, 1.7 H), 3.54 (s, 1.3 H), 1.27 (d, J = 6.8 Hz, 1.3 H), 1.19
(d, J = 6.8 Hz, 1.7 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.7, 162.6 (t, J = 28.9 Hz),
162.5 (t, J = 28.9 Hz), 146.6, 146.5, 135.5, 135.5, 135.1, 135.1, 128.6,
128.6, 128.0, 117.1, 117.0 115.9 (dd, J = 257.2, 256.2 Hz), 115.8 (dd, J =
257.2, 254.3 Hz), 113.4, 58.1 (t, J = 21.2 Hz), 57.9 (t, J = 22.2 Hz), 51.8,
51.8, 47.7, 47.6, 16.3.
Note: All aromatic carbons could not be observed in 13C NMR spectra
of the products 3a–v and 6 reported below, probably due to overlap-
ping.
19F NMR (DMSO-d6, 376 MHz):  = –107.5 (dd, J = 251.4, 9.4 Hz, 0.43
F), –108.9 (dd, J = 250.3, 10.5 Hz, 0.59 F), –115.9 (dd, J = 250.7, 18.8
Hz, 0.58 F), –117.1 (dd, J = 251.1, 19.9 Hz, 0.42 F).
MS (EI): m/z = 362 [M]+.
HRMS (EI): m/z [M]+ calcd for C19H20F2N2O3: 362.1442; found:
,-Difluoro--lactams 2a,b; General Procedure
Ethyl bromodifluoroacetate (20 mmol) and the corresponding imine
(10 mmol) were added to a solution of RhCl(PPh3)3 (1 mol%) in THF
(60 mL) at 0 °C and stirred for 0.5 h. A 1.0 M solution of Et2Zn in hex-
ane (30 mmol) was slowly added to the solution via a dropping fun-
nel, and then the whole mixture was stirred at the same temperature.
The reaction was quenched with aq 10% HCl, the mixture was extract-
ed with EtOAc, and the combined extracts were washed with brine
and dried (MgSO4). The solvent was removed in vacuo and the residue
was purified by flash column chromatography to give the correspond-
ing ,-difluoro--lactams 2a,b.
362.1446.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-valinate (3c)
Eluent: EtOAc/hexane (3:7); yield: 56.6 mg (72%); dr 1:1.2; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.83 (d, J = 8.2 Hz, 0.54 H), 8.66 (d,
J = 8.4 Hz, 0.46 H), 7.55–7.52 (m, 2 H), 7.35–7.26 (m, 3 H), 7.03–7.00
(m, 2 H), 6.75–6.70 (m, 2 H), 6.56–6.53 (m, 1 H), 6.35 (d, J = 11.0 Hz,
0.47 H), 6.34 (d, J = 10.4 Hz, 0.56 H), 5.40 (ddd, J = 19.7, 10.4, 9.7 Hz,
0.56 H), 5.30 (ddd, J = 22.2, 11.0, 7.3 Hz, 0.47 H), 4.21 (dd, J = 8.4, 7.8
Hz, 0.46 H), 4.17 (dd, J = 8.2, 7.8 Hz, 0.54 H), 3.6 (s, 1.56 H), 3.57 (s,
1.32 H), 2.16–1.98 (m, 1 H), 0.85 (dd, J = 15.6, 6.9 Hz, 2.9 H), 0.74 (dd,
J = 11.4, 6.9 Hz, 3.3 H).
Ring-Opening Peptide Synthesis; General Procedure
To a vial containing difluoro--lactam 2a (51.9 mg, 0.2 mmol) and
amino acid ester hydrochloride (0.4 mmol) in TFE (0.3 mL) was added
DIPEA (0.10 mL, 0.6 mmol) at r.t. and the mixture was stirred at r.t. for
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

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A. Tarui et al.
Paper
Synthesis
13C{1H} NMR (DMSO-d6, 100 MHz):  = 170.9, 170.8, 163.1 (t, J = 28.9
Hz), 162.9 (t, J = 29.5 Hz), 146.5, 146.3, 135.3, 135.1, 128.7, 128.6,
128.5, 127.9, 127.9, 117.1, 117.0, 115.9 (dd, J = 256.7, 253.3 Hz), 115.8
(dd, J = 256.2, 255.3 Hz), 113.5, 113.4, 58.2–57.5 (m), 57.8, 51.7, 29.7,
29.5, 18.7, 18.6, 18.2, 18.1.
19F NMR (DMSO-d6, 376 MHz):  = –106.2 (dd, J = 250.7, 7.3 Hz, 0.47
F), –107.9 (dd, J = 251.4, 9.7 Hz, 0.56 F), –115.3 (dd, J = 251.4, 19.7 Hz,
0.56 F), –117.5 (dd, J = 250.7, 22.2 Hz, 0.47 F).
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-serinate (3f)
Eluent: EtOAc/hexane (1:1); yield: 26.3 mg (35%); dr 1:1; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.77–8.75 (m, 1 H), 7.59–7.56 (m, 2
H), 7.40–7.31 (m, 3 H), 7.08–7.04 (m, 2 H), 6.79–6.76 (m, 2 H), 6.61–
6.57 (m, 1 H), 6.36–6.33 (m, 1 H), 5.42–5.30 (m, 1 H), 5.10–5.04 (m, 1
H), 4.46–4.39 (m, 1 H), 3.80–3.63 (m, 2 H), 3.61 (s, 1.5 H), 3.59 (s, 1.5
H).
MS (EI): m/z = 390 [M]+.
13C{1H} NMR (DMSO-d6, 100 MHz):  = 169.9, 169.8, 163.2–162.6 (m),
146.6, 146.5, 135.2, 135.1, 128.7, 128.6, 128.6, 128.0, 117.2, 117.1,
115.9 (dd, J = 258.6, 254.4 Hz), 115.8 (dd, J = 259.1, 254.3 Hz), 113.6,
113.5, 60.7, 58.1 (dd, J = 27.2, 21.7 Hz), 57.9 (dd, J = 26.7, 21.8 Hz),
54.9, 54.8, 51.9.
19F NMR (DMSO-d6, 376 MHz):  = –107.1 (dd, J = 251.4, 8.7 Hz, 0.5 F),
–107.3 (dd, J = 251.9, 9.4 Hz, 0.5 F), –116.7 (dd, J = 251.4, 19.7 Hz, 0.5
F), –117.1 (dd, J = 251.9, 20. 3 Hz, 0.5 F).
HRMS (EI): m/z [M]+ calcd for C21H24F2N2O3: 390.1755; found:
390.1758.
Methyl [2,2-difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-leucinate (3d)
Eluent: EtOAc/hexane (1:4); yield: 57.0 mg (70%); dr 1:1; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.98 (d, J = 8.2 Hz, 0.5 H), 8.91 (d, J =
7.8 Hz, 0.5 H), 7.53–7.51 (m, 2 H), 7.36–7.27 (m, 3 H), 7.04–7.00 (m, 2
H), 6.72–6.70 (m, 2 H), 6.56–6.53 (m, 1 H), 6.34 (d, J = 10.5 Hz, 0.5 H),
6.27 (d, J = 10.5 Hz, 0.5 H), 5.37–5.22 (m, 1 H), 4.38–4.28 (m, 1 H), 3.60
(s, 1.5 H), 3.56 (s, 1.5 H), 1.72–1.20 (m, 3 H), 0.80 (d, J = 6.0 Hz, 1.5 H),
0.68 (d, J = 6.4 Hz, 3 H), 0.62 (d, J = 6.4 Hz, 1.5 H).
13C{1H} NMR (acetone-d6, 100 MHz):  = 172.8, 172.7, 164.2 (t, J = 28.5
Hz), 164.0 (t, J = 28.5 Hz), 147.6, 147.3, 136.3, 136.2, 136.1, 129.7,
129.6, 129.2, 129.1, 129.1, 118.7, 118.6, 117.2 (dd, J = 260.9, 257.0
Hz), 117.0 (dd, J = 260.6, 254.8 Hz), 114.6, 60.1 (dd, J = 25.5, 22.1 Hz),
59.7 (dd, J = 27.9, 22.1 Hz), 52.5, 52.5, 51.5, 51.4, 40.9, 40.7, 25.0, 24.9,
23.3, 23.2, 21.5, 21.3.
MS (EI): m/z = 378 [M]+.
HRMS (EI): m/z [M]+ calcd for C19H20F2N2O4: 378.1391; found:
378.1389.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-threoninate (3g)
Eluent: EtOAc/hexane (3:2); yield: 35.0 mg (45%); dr 1:1; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.39 (d, J = 8.7 Hz, 0.5 H), 8.26 (d, J =
8.2 Hz, 0.5 H), 7.61–7.59 (m, 2 H), 7.42–7.33 (m, 3 H), 7.08–7.05 (m, 2
H), 6.81–6.76 (m, 2 H), 6.61–6.58 (m, 1 H), 6.46–6.42 (m, 1 H), 5.48–
5.32 (m, 1 H), 5.12 (d, J = 6.4 Hz, 0.5 H), 5.02 (d, J = 6.4 Hz, 0.5 H), 4.40
(dd, J = 14.4, 8.4 Hz, 0.5 H), 4.39 (dd, J = 14.1, 8.7 Hz, 0.5 H), 4.25–4.13
(m, 1 H), 3.65 (s, 1.5 H), 3.64 (s, 1.5 H), 1.10 (d, J = 6.4 Hz, 1.5 H), 0.92
(d, J = 6.4 Hz, 1.5 H).
19F NMR (DMSO-d6, 376 MHz):  = –106.7 (dd, J = 250.3, 8.3 Hz, 0.5 F),
–109.4 (dd, J = 250.7, 10.8 Hz, 0.5 F), –115.0 (dd, J = 250.7, 18.8 Hz, 0.5
F), –117.9 (dd, J = 250.3, 21.3 Hz, 0.5 F).
MS (EI): m/z = 404 [M]+.
13C{1H} NMR (DMSO-d6, 100 MHz):  = 169.9, 169.8, 163.2 (t, J = 28.9
Hz), 146.6, 146.4, 135.2, 135.2, 135.0, 128.7, 128.6, 128.6, 127.9,
117.1, 117.0, 116.1 (dd, J = 257.7, 254.8 Hz), 116.0 (dd, J = 260.6, 255.7
Hz), 113.6, 66.0, 65.9, 58.0, 57.9 (dd, J = 26.0, 21.2 Hz), 57.6 (dd, J =
28.4, 22.2 Hz), 51.9, 51.9, 19.7, 19.5.
19F NMR (DMSO-d6, 376 MHz):  = –105.60 (dd, J = 250.3, 6.9 Hz, 0.5
F), –108.45 (dd, J = 251.4, 10.5 Hz, 0.5 F), –115.47 (dd, J = 251.4, 19.5
Hz, 0.5 F), –118.66 (dd, J = 250.3, 22.8 Hz, 0.5 F).
HRMS (EI): m/z [M]+ calcd for C22H26F2N2O3: 404.1911; found:
404.1912.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-isoleucinate (3e)
Eluent: EtOAc/hexane (3:7); yield: 65.6 mg (81%); dr 1:1.1; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.86 (d, J = 8.2 Hz, 0.5 H), 8.67 (d, J =
8.2 Hz, 0.42 H), 7.54–7.51 (m, 2 H), 7.35–7.28 (m, 3 H), 7.03–6.99 (m,
2 H), 6.73–6.69 (m, 2 H), 6.56–6.52 (m, 1 H), 6.37–6.32 (m, 1 H), 5.34–
5.45 (m, 1 H), 5.40 (ddd, J = 19.8, 10.8, 9.5 Hz, 0.52 H), 5.28 (ddd, J =
21.9, 11.0, 7.8 Hz, 0.48 H), 4.25–4.16 (m, 1 H), 3.61 (s, 1.48 H), 3.57 (s,
1.36 H), 1.89–1.74 (m, 1 H), 1.45–0.91 (m, 2 H), 0.81–0.62 (m, 6 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.1, 170.9, 162.9 (t, J = 29.4
Hz), 162.8 (t, J = 29.4 Hz), 146.5, 146.3, 135.3, 135.1, 128.7, 128.6,
128.6, 128.5, 128.0, 127.9, 117.1, 117.0, 115.9 (dd, J = 259.1, 253.4
Hz), 115.8 (dd, J = 257.2, 255.3 Hz), 113.4, 113.4, 57.8 (dd, J = 27.9,
22.1 Hz), 57.7 (dd, J = 28.4, 21.2 Hz), 56.7, 56.6, 51.7, 39.50, 35.8, 35.7,
24.5, 24.4, 15.0, 14.9, 10.6, 10.3.
19F NMR (DMSO-d6, 376 MHz):  = –106.4 (dd, J = 250.3, 7.8 Hz, 0.48
F), –107.8 (dd, J = 251.4, 9.5 Hz, 0.52 F), –115.5 (dd, J = 251.1, 19.8 Hz,
0.52 F), –117.5 (dd, J = 250.3, 21.9 Hz, 0.48 F).
MS (EI): m/z = 404 [M]+.
HRMS (EI): m/z [M]+ calcd for C22H26F2N2O3: 404.1911; found:
MS (EI): m/z = 392 [M]+.
HRMS (EI): m/z [M]+ calcd for C20H22F2N2O4: 392.1548; found:
392.1549.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-tyrosinate (3h)
Eluent: EtOAc/hexane (3:7); yield: 75.1 mg (83%); dr 1:1; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 9.13 (br s, 0.5 H), 9.12 (br s, 0.5 H),
8.96 (d, J = 7.3 Hz, 0.5 H), 8.94 (d, J = 7.3 Hz, 0.5 H), 7.50–7.28 (m, 5 H),
7.05–6.88 (m, 4 H), 6.72–6.54 (m, 5 H), 6.26 (d, J = 9.2 Hz, 0.5 H), 6.23
(d, J = 8.6 Hz, 0.5 H), 5.28 (ddd, J = 18.8, 10.5, 8.6 Hz, 1 H), 5.21 (ddd,
J = 19.5, 9.2, 8.3 Hz, 1 H), 4.43–4.37 (m, 1 H), 3.54 (s, 1.5 H), 3.50 (s, 1.5
H), 3.00–2.81 (m, 2 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 170.9, 170.8, 162.8 (t, J = 28.9
Hz), 162.7 (t, J = 28.9 Hz), 156.0, 155.9, 146.6, 146.5, 135.2, 135.1,
129.9, 129.8, 128.7, 128.7, 128.6, 128.0, 127.9, 126.9, 126.8, 117.2,
404.1919.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

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117.1, 115.8 (dd, J = 259.1, 254.3 Hz), 115.7 (dd, J = 258.7, 254.5 Hz),
115.1, 115.0, 113.6, 113.5, 58.0 (dd, J = 27.0, 22.2 Hz), 54.2, 54.1, 51.8,
51.8, 35.3, 35.2.
19F NMR (DMSO-d6, 376 MHz):  = –106.9 (dd, J = 253.2, 8.3 Hz, 0.5 F),
–108.3 (dd, J = 251.1, 10.5 Hz, 0.5 F), –115.9 (dd, J = 251. 4, 18.8 Hz, 0.5
F), –116.4 (dd, J = 252.9, 19.5 Hz, 0.5 F).
1H NMR (DMSO-d6, 400 MHz):  = 8.9 (d, J = 7.8 Hz, 0.5 H), 8.8 (d, J =
8.2 Hz, 0.5 H), 7.5 (m, 2 H), 7.4–7.3 (m, 3 H), 7.0–6.9 (m, 2 H), 6.7 (m, 2
H), 6.6 (br s, 1 H), 6.5 (m, 2 H), 6.3 (m, 1 H), 5.4–5.2 (m, 1 H), 4.3–4.2
(m, 1 H), 3.6 (s, 1.5 H), 3.5 (s, 1.5 H), 2.9–2.7 (m, 2 H), 1.8–1.5 (m, 2 H),
1.4 (s, 9 H), 1.3–0.9 (m, 4 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.4, 171.3, 162.9 (t, J = 28.9
Hz), 162.9 (t, J = 28.9 Hz), 155.4, 146.6, 146.4, 135.2, 135.2, 135.1,
128.7, 128.6, 128.0, 117.1, 117.1, 115.9 (dd, J = 257.5, 256.7 Hz), 115.8
(dd, J = 261.0, 253.4 Hz), 113.4, 77.2, 58.0 (dd, J = 26.4, 21.7 Hz), 57.8
(dd, J = 27.8, 21.5 Hz), 52.1, 52.0, 51.8, 51.8, 39.2,a,b 29.9,a 29.9,a 28.9,a
28.7,a 28.1, 22.4,a 22.3.a a These signals were observed as reverse im-
age in DEPT 135. b This signal overlapped with solvent peaks (DMSO-
d6).
MS (EI): m/z = 454 [M]+.
HRMS (EI): m/z [M]+ calcd for C25H24F2N2O4: 454.1704; found:
454.1705.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-phenylalaninate (3i)
Eluent: EtOAc/hexane (3:7); yield: 64.9 mg (74%); dr 1:1; colorless
solid.
19F NMR (DMSO-d6, 376 MHz):  = –107.0 (dd, J = 250.5, 6.1 Hz, 0.5 F),
–109.4 (dd, J = 251.2, 9.4 Hz, 0.5 F), –115.1 (dd, J = 251.2, 17.7 Hz, 0.5
F), –117.3 (dd, J = 250.5, 21.0 Hz, 0.5 F).
MS (EI): m/z = 519 [M]+.
HRMS (EI): m/z [M]+ calcd for C27H35F2N3O5: 519.2545; found:
1H NMR (DMSO-d6, 400 MHz):  = 9.11 (d, J = 7.8 Hz, 1 H), 7.55–7.06
(m, 12 H), 6.78–6.60 (m, 3 H), 6.30 (d, J = 10.6 Hz, 0.5 H), 6.28 (d, J =
10. 3 Hz, 0.5 H), 5.33 (ddd, J = 20.2, 10.6, 8.7 Hz, 0.5 H), 5.26 (ddd, J =
18.8, 10.3, 10.1 Hz, 0.5 H), 4.57–4.51 (m, 1 H), 3.60 (s, 1.5 H), 3.55 (s,
1.5 H), 3.10 (dd, J = 14.0, 5.3 Hz, 0.5 H), 3.06 (dd, J = 13.9, 5.2 Hz, 0.5
H), 2.99 (dd, J = 14.0, 9.3 Hz, 0.5 H), 2.95 (dd, J = 13.9, 9.1 Hz, 0.5 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 170.7, 162.8 (t, J = 28.9 Hz),
162.8 (t, J = 29.5 Hz), 146.5, 136.9, 136.8, 135.1, 135.0, 128.9, 128.8,
128.6, 128.6, 128.5, 128.1, 128.0, 127.9, 127.9, 126.4, 126.3, 117.1,
117.1, 115.8 (dd, J = 260.1, 254.3 Hz), 115.6 (dd, J = 260.1, 254.3 Hz),
113.6, 113.5, 57.9 (dd, J = 27.0, 22.1 Hz), 57.9 (dd, J = 28.3, 22.1 Hz),
53.8, 53.7, 51.9, 51.8, 35.9, 35.8.
19F NMR (DMSO-d6, 376 MHz):  = –106.67 (dd, J = 253.6, 8.7 Hz, 0.5
F), –107.94 (dd, J = 251.4, 10.1 Hz, 0.5 F), –116.3 (dd, J = 251.4, 18.8
Hz, 0.5 F), –116.6 (dd, J = 253.6, 20.2 Hz, 0.5 F).
MS (EI): m/z = 438 [M]+.
HRMS (EI): m/z [M]+ calcd for C25H24F2N2O3: 438.1755; found:
519.2546.
Methyl N2-[2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
N-nitro-L-argininate (3l)
Amino acid (1 equiv) and DIPEA (2 equiv) were used; eluent: EtO-
Ac/hexane (4:1); yield: 80.5 mg (82%); dr 1:1.2; colorless solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.9 (m, 1 H), 8.4 (s, 1 H), 7.9 (s, 2 H),
7.5 (m, 2 H), 7.4–7.3 (m, 3 H), 7.0 (m, 2 H), 6.7–6.5 (m, 3 H), 6.3 (m, 1
H), 5.3–5.2 (m, 1 H), 4.3–4.2 (m, 1 H), 3.6 (s, 1.6 H), 3.5 (s, 1.4 H), 3.1–
2.9 (m, 2 H), 1.9–1.2 (m, 4 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.2, 171.1, 163.0 (t, J = 28.9
Hz), 162.9 (t, J = 29.5 Hz), 159.2, 159.2, 146.6, 146.4, 135.2, 135.2,
135.1, 135.1, 128.7, 128.6, 128.0, 117.1, 117.1, 116.0 (dd, J = 253.3,
248.1 Hz), 113.4, 113.4, 58.3–57.6 (m), 51.9, 51.9, 51.8, 39.9,a,b 27.4,a
27.3,a 24.6.a a These signals were observed as reverse image in DEPT
135. b This signal overlapped with solvent peaks (DMSO-d6).
19F NMR (DMSO-d6, 376 MHz):  = –107.2 (d, J = 250.4 Hz, 0.4 F),
–109.7 (dd, J = 249.5, 10.8 Hz, 0.6 F), –114.9 (dd, J = 249.5, 17.7 Hz, 0.6
F), –117.1 (dd, J = 250.4, 20.2 Hz, 0.4 F).
438.1764.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-tryptophanate (3j)
Eluent: EtOAc/hexane (3:7); yield: 104.2 mg (73%); dr 1:1; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 10.8 (s, 0.5 H), 10.7 (s, 0.5 H), 9.0 (d,
J = 7.3 Hz, 0.5 H), 8.9 (d, J = 7.8 Hz, 0.5 H), 7.5–6.9 (m, 12 H), 6.7–6.5
(m, 3 H), 6.3 (d, J = 10.5 Hz, 1 H), 5.2–5.3 (m, 1 H), 4.5 (m, 1 H), 3.5 (s,
1.5 H), 3.5 (s, 1.5 H), 3.3–3.1 (m, 2 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.1, 171.0, 162.7 (t, J = 29.0
Hz), 146.6, 146.5, 136.1, 136.0, 135.2, 135.1, 135.0, 135.0, 128.6,
128.5, 128.0, 127.9, 127.9, 126.9, 126.8, 123.6, 123.4, 120.9, 120.9,
118.4, 118.3, 117.8, 117.7, 117.1, 117.1, 115.8 (dd, J = 259.2, 254.9
Hz), 115.8 (dd, J = 259.2, 255.0 Hz), 113.5, 113.4, 111.4, 111.3, 109.1,
109.0, 58.1 (dd, J = 26.7, 21.9 Hz), 53.3, 53.2, 51.8, 51.8, 26.4, 26.3.
MS (FAB): m/z = 493 [M + H]+.
HRMS (FAB): m/z [M + H]+ calcd for C22H27F2N6O5: 493.2011; found:
493.2014.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-histidinate (3m)
The reaction was carried out on a 0.3 mmol scale. Histidine methyl
ester 2 HCl (2 equiv) and DIPEA (5 equiv) were used; eluent: EtO-
Ac/hexane (3:7) → EtOH/CHCl3 (5:95); yield: 73.3 mg (80%); dr 1:1.1;
colorless solid.
1H NMR (DMSO-d6, 400 MHz):  = 11.8 (s, 1 H), 9.1 (d, J = 7.8 Hz, 0.5
H), 9.1 (d, J = 7.8 Hz, 0.5 H), 7.6–7.3 (m, 6 H), 7.0 (m, 2 H), 6.8 (s, 0.5 H),
6.7 (m, 2.5 H), 6.5 (m, 1 H), 6.3 (m, 1 H), 5.3–5.2 (m, 1 H), 4.6–4.4 (m, 1
H), 3.51 (s, 1.5 H), 3.5 (s, 1.5 H), 2.9 (d, J = 6.4 Hz, 2 H), 2.9 (d, J = 6.4
Hz, 2 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 170.6, 162.7 (t, J = 28.9 Hz),
162.6 (t, J = 28.6 Hz), 146.6, 146.5, 135.2, 135.1, 135.0, 134.9, 134.9,
128.6, 128.6, 128.6, 128.6, 127.9, 117.1, 117.0, 115.8 (dd, J = 259.6,
256.3 Hz), 113.5, 58.1 (dd, J = 27.0, 23.1 Hz), 57.9 (dd, J = 27.0, 23.1
Hz), 52.5, 52.4, 51.8, 28.4.
19F NMR (DMSO-d6, 376 MHz):  = –107.98 (dd, J = 251.4, 10.1 Hz, 0.5
F), –109.19 (dd, J = 250.7, 10.8 Hz, 0.5 F), –115.3 (dd, J = 250.3, 18.3
Hz, 0.5 F), –116.0 (dd, J = 251.3, 19.2 Hz, 0.5 F).
MS (EI): m/z = 477 [M]+.
HRMS (EI): m/z [M]+ calcd for C27H25F2N3O3: 477.1864; found:
477.1868.
Methyl N6-(tert-Butoxycarbonyl)-N2-[2,2-difluoro-3-phenyl-
3-(phenylamino)propanoyl]-L-lysinate (3k)
Eluent: EtOAc/hexane (3:7); yield: 76.2 mg (73%); dr 1:1; colorless
solid.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

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A. Tarui et al.
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19F NMR (DMSO-d6, 376 MHz):  = –107.3 (dd, J = 252.1, 8.7 Hz, 0.47
F), –108.1 (dd, J = 251.8, 9.2 Hz, 0.53 F), –116.34 (dd, J = 251.8, 18.8
Hz, 0.53 F), –117.21 (dd, J = 252.1, 19.9 Hz, 0.47 F).
MS (EI): m/z = 428 [M]+.
HRMS (EI): m/z [M]+ calcd for C22H22F2N4O3: 428.1660; found:
1H NMR (DMSO-d6, 400 MHz):  = 9.0 (d, J = 8.2 Hz, 0.52 H), 8.9 (d, J =
8.2 Hz, 0.48 H), 7.5 (m, 2 H), 7.4–7.3 (m, 3 H), 7.0 (m, 2 H), 6.7 (m, 2
H), 6.5 (m, 1 H), 6.3 (d, J = 10.6 Hz, 0.52 H), 6.3 (d, J = 10.6 Hz, 0.48 H),
5.4–5.2 (m, 1 H), 4.4–4.3 (m, 1 H), 3.6 (s, 1.57 H), 3.6 (s, 1.43 H), 2.3–
1.7 (m, 4 H), 1.3 (s, 4.71 H), 1.3 (s, 4.28 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.2, 171.2, 171.0, 170.9,
162.9 (t, J = 28.9 Hz), 162.9 (t, J = 29.0 Hz), 146.6, 146.4, 135.2, 135.2,
135.0, 128.7, 128.7, 128.6, 128.0, 117.1, 117.1, 115.9 (dd, J = 261.0,
255.9 Hz), 115.9 (dd, J = 261.4, 253.4 Hz), 113.5, 113.4, 79.6, 58.1 (dd,
J = 26.0, 22.2 Hz), 57.9 (dd, J = 28.2, 21.9 Hz), 51.9, 51.92, 5.17, 30.7,
30.6, 27.6, 25.6, 25.5.
19F NMR (DMSO-d6, 376 MHz):  = –106.8 (dd, J = 251.3, 8.3 Hz, 0.48
F), –109.5 (dd, J = 250.7, 10.8 Hz, 0.52 F), –115.0 (dd, J = 250.7, 18.1
Hz, 0.52 F), –117.7 (dd, J = 251.3, 21.0 Hz, 0.48 F).
MS (EI): m/z = 476 [M]+.
HRMS (EI): m/z [M]+ calcd for C25H30F2N2O5: 476.2123; found:
428.1668.
Di-tert-butyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-aspartate (3n)
Eluent: EtOAc/hexane (1:9); yield: 58.0 mg (57%); dr 1:1.1; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.9 (m, 1 H), 7.5 (m, 2 H), 7.3–7.2
(m, 3 H), 7.0 (m, 2 H), 6.7 (m, 2 H), 6.5 (m, 1 H), 6.3 (d, J = 11.0 Hz, 1
H), 5.3–5.2 (m, 1 H), 4.5 (dd, J = 15.0, 6.7 Hz, 0.5 H), 4.5 (dd, J = 13.8,
6.5 Hz, 0.5 H), 2.7–2.6 (m, 1 H), 2.5–2.4 (m, 1 H),a 1.3 (s, 4.5 H), 1.3 (s,
a
4.5 H), 1.3 (s, 4.5 H), 1.3 (s, 4.5 H). This signal overlapped with sol-
476.2124.
vent peaks (DMSO-d6).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 168.8, 168.7, 168.6, 162.6 (t,
J = 28.9 Hz), 162.5 (t, J = 28.9 Hz), 146.6, 146.5, 135.2, 135.1, 128.7,
128.5, 128.0, 117.19, 115.8 (dd, J = 260.1, 254.3 Hz), 115.8 (dd, J =
259.6, 254.7 Hz), 113.7, 113.6, 81.3, 81.3, 80.5, 80.4, 58.2–57.5 (m),
49.4, 49.3, 36.4, 36.3, 27.5, 27.5, 27.3, 27.3.
tert-Butyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-glutaminate (3q)
Eluent: EtOAc/hexane (3:2): yield: 72.2 mg (76%); dr 1:1; colorless
solid.
19F NMR (DMSO-d6, 376 MHz):  = –106.5 (dd, J = 253.2, 8.3 Hz, 0.48
F), –107.6 (dd, J = 252.1, 9.4 Hz, 0.52 F), –116.9 (dd, J = 252.1, 19.5 Hz,
0.52 F), –117.5 (dd, J = 253.2, 20.9 Hz, 0.48 F).
MS (EI): m/z = 504 [M]+.
HRMS (EI): m/z [M]+ calcd for C27H34F2N2O5: 504.2436; found:
1H NMR (DMSO-d6, 400 MHz):  = 8.9 (d, J = 7.1 Hz, 0.5 H), 8.9 (d, J =
7.1 Hz, 0.5 H), 7.5 (m, 2 H), 7.4–7.2 (m, 3 H), 7.1–7.0 (m, 3 H), 6.7 (m, 3
H), 6.5 (m, 1 H), 6.3 (d, J = 10.4 Hz, 1 H), 6.3 (d, J = 10.5 Hz, 1 H), 5.3–
5.2 (m, 1 H), 4.1–4.0 (m, 1 H), 2.2–1.7 (m, 4 H), 1.3 (s, 4.5 H), 1.3 (s, 4.5
H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 173.3, 173.3, 169.8, 169.8,
162.8 (t, J = 28.9 Hz), 146.6, 146.4, 135.2, 135.2, 135.1, 128.7, 128.6,
128.6, 128.0, 117.1, 117.1, 115.8 (dd, J = 257.7, 255.5 Hz), 115.8 (dd,
J = 260.6, 253.7 Hz), 113.6, 80.8, 58.1 (dd, J = 26.8, 22.2 Hz), 57.8 (dd,
J = 28.0, 21.8 Hz), 52.8, 30.9, 30.9, 27.4, 25.9, 25.9.
19F NMR (DMSO-d6, 376 MHz):  = –106.5 (dd, J = 252.7, 8.3 Hz, 0.5 F),
–108.47 (dd, J = 251.6, 10.5 Hz, 0.5 F), –115.8 (dd, J = 251.6, 18.8 Hz,
0.5 F), –117.3 (dd, J = 252.7, 20.2 Hz, 0.5 F).
MS (EI): m/z = 461 [M]+.
HRMS (EI): m/z [M]+ calcd for C24H29F2N3O4: 461.2126; found:
504.2439.
tert-Butyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-asparaginate (3o)
Eluent: MeOH/CHCl3 (5:95); yield: 28.8 mg (31%); dr 1:1.2; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 8.7 (m, 1 H), 7.4 (m, 2 H), 7.3–7.2
(m, 4 H), 6.9 (m, 2 H), 6.8 (br d, J = 21.5 Hz, 1 H), 6.6 (m, 2 H), 6.5 (m, 1
H), 6.2 (d, J = 10.6 Hz, 0.45 H), 6.2 (d, J = 10.4 Hz, 0.55 H), 5.2 (ddd, J =
20.2, 10.4, 9.6 Hz, 1 H), 4.5–4.4 (m, 1 H), 2.5–2.3 (m, 2 H),a 1.3 (s, 9 H).
a This signal overlapped with solvent peaks (DMSO-d6).
461.2131.
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.1, 171.0, 169.1, 162.4 (t,
J = 28.9 Hz), 162.3 (t, J = 28.9 Hz), 146.6, 146.5, 135.2, 135.1, 128.7,
128.7, 128.6, 128.0, 117.2, 115.9 (dd, J = 259.1, 255.3 Hz), 115.9 (dd,
J = 260.6, 253.8 Hz), 113.7, 113.6, 80.9, 58.1 (dd, J = 26.7, 22.3 Hz),
57.8 (dd, J = 28.0, 21.8 Hz), 49.6, 49.5, 35.9, 35.9, 27.4.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-cysteinate (3r)
Eluent: EtOAc/hexane (1:4); yield: 58.1 mg (74%); dr 1:1.1; colorless
solid.
19F NMR (DMSO-d6, 376 MHz):  = –106.6 (dd, J = 252.3, 8.7 Hz, 0.45
F), –108.0 (dd, J = 251.3, 10.1 Hz, 0.55 F), –116.5 (dd, J = 251.3, 19.1
Hz, 0.55 F), –117.8 (dd, J = 252.3, 20.6 Hz, 0.45 F).
MS (EI): m/z = 447 [M]+.
HRMS (EI): m/z [M]+ calcd for C23H27F2N3O4: 447.1970; found:
1H NMR (DMSO-d6, 400 MHz):  = 9.1 (d, J = 8.2 Hz, 0.48 H), 9.0 (d, J =
7.8 Hz, 0.52 H), 7.5 (m, 2 H), 7.4–7.3 (m, 3 H), 7.0 (m, 2 H), 6.7 (m, 2
H), 6.5 (m, 1 H), 6.3 (d, J = 10.5 Hz, 0.52 H), 6.3 (d, J = 10.4 Hz, 0.48 H),
5.4–5.2 (m, 1 H), 4.5–4.4 (m, 1 H), 3.5 (s, 1.44 H), 3.5 (s, 1.56 H), 2.9–
2.7 (m, 2 H), 2.4 (t, J = 8.5 Hz, 0.52 H), 2.2 (t, J = 8.7 Hz, 0.48 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 169.6, 162.9 (t, J = 28.9 Hz),
162.9 (t, J = 29.8 Hz), 146.6, 146.4, 135.1, 135.0, 128.7, 128.6, 128.0,
117.2, 117.2, 115.9 (dd, J = 260.6, 254.2 Hz), 115.9 (dd, J = 257.4, 257.4
Hz), 113.5, 58.0 (dd, J = 27.1, 21.2 Hz), 57.8 (dd, J = 26.8, 21.1 Hz), 54.9,
54.8, 52.1, 24.5, 24.5.
447.1974.
5-tert-Butyl 1-Methyl [2,2-Difluoro-3-phenyl-3-(phenyl-
amino)propanoyl]-L-glutamate (3p)
Eluent: EtOAc/hexane (1:4); yield: 72.4 mg (76%); dr 1:1.1; colorless
solid.
19F NMR (DMSO-d6, 376 MHz):  = –107.1 (dd, J = 251.2, 8.7 Hz, 0.52
F), –108.2 (dd, J = 251.8, 9.8 Hz, 0.48 F), –116.0 (dd, J = 251.8, 18.8 Hz,
0.48 F), –117.4 (dd, J = 251.2, 20.6 Hz, 0.52 F).
MS (EI): m/z = 394 [M]+.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

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A. Tarui et al.
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Synthesis
HRMS (EI): m/z [M]+ calcd for C19H20F2N2O3S: 394.1163; found:
394.1158.
19F NMR (DMSO-d6, 376 MHz):  = –106.6 (dd, J = 251.2, 8.7 Hz, 1 F),
–117.8 (dd, J = 251.2, 20.6 Hz, 1 F).
MS (EI): m/z = 462 [M]+.
HRMS (EI): m/z [M]+ calcd for C22H24F2N4O5: 462.1715; found:
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
L-methioninate (3s)
462.1716.
Eluent: EtOAc/hexane (3:7); yield: 60.9 mg (71%); dr 1:1.1; colorless
solid.
Peptide Elongation Procedure for the Synthesis of Tripeptides 6
Methyl (3-Amino-2,2-difluoro-3-phenylpropanoyl)glycinate (5)
1H NMR (DMSO-d6, 400 MHz):  = 9.0 (d, J = 7.8 Hz, 0.52 H), 8.9 (d, J =
7.8 Hz, 0.48 H), 7.5 (m, 2 H), 7.4–7.3 (m, 3 H), 7.0 (m, 2 H), 6.7 (m, 2
H), 6.5 (m, 1 H), 6.3 (d, J = 10.7 Hz, 0.52 H), 6.3 (d, J = 10.8 Hz, 0.48 H),
5.4–5.2 (m, 1 H), 4.5–4.4 (m, 1 H), 3.6 (s, 1.57 H), 3.6 (s, 1.43 H), 2.4–
1.8 (m, 7 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 171.1, 171.0, 163.0 (t, J = 28.9
Hz), 162.9 (t, J = 29.8 Hz), 146.6, 146.4, 135.2, 135.2, 135.0, 128.7,
128.7, 128.6, 128.0, 117.2, 117.1, 115.9 (dd, J = 255.3, 254.3 Hz), 115.9
(dd, J = 261.0, 256.4 Hz), 113.4, 113.4, 58.2–57.6 (m), 52.0, 52.0, 50.9,
50.9, 29.9, 29.8, 29.3, 29.2, 14.3, 14.2.
19F NMR (DMSO-d6, 376 MHz):  = –106.7 (dd, J = 251.1, 7.9 Hz, 0.48
F), –109.4 (dd, J = 250.7, 10.8 Hz, 0.52 F), –115.1 (dd, J = 250.7, 18.8
Hz, 0.52 F), –117.9 (dd, J = 251.1, 21.3 Hz, 0.48 F).
MS (EI): m/z = 422 [M]+.
HRMS (EI): m/z [M]+ calcd for C21H24F2N2O3S: 422.1476; found:
To a vial containing difluoro--lactam 2b (145 mg, 0.5 mmol) and
glycine methyl ester hydrochloride (126 mg, 1.0 mmol) in TFE (2.5
mL) was added DIPEA (0.26 mL, 1.5 mmol) at r.t. Then, the mixture
was stirred at r.t. for 2 h, poured into H2O, and extracted with EtOAc.
The combined organic phases were washed with brine, dried
(MgSO4), and concentrated in vacuo. The crude fluorinated dipeptide
4 was used to the oxidative deprotection reactions without further
purification. To a vial containing the crude dipeptide 4 in MeCN (10
mL) was added a solution of ceric ammonium nitrate (548 mg, 1.0
mmol) in H2O (1.1 mL) at 0 °C over 10 min under air. The mixture was
stirred at r.t. for 2 h. The mixture was poured into sat. aq NaHCO3, and
extracted with EtOAc. The combined organic phases were washed
with sat. aq Na2SO3 (2 ×) and brine, dried (MgSO4), and concentrated
in vacuo. The crude mixture was purified by flash column chromatog-
raphy (70% EtOAc in hexane) to give the pure product 5 as a colorless
liquid; yield: 77 mg (70% based on 2b).
422.1475.
Methyl (S)-2-[2,2-Difluoro-3-phenyl-3-(phenylamino)propanami-
do]-2-phenylacetate (3u)
1H NMR (CDCl3, 400 MHz):  = 7.4–7.3 (m, 5 H), 7.1 (br s, 1 H), 4.6 (dd,
J = 16.3, 10.2 Hz, 1 H), 4.1 (dd, J = 18.3, 5.3 Hz, 1 H), 3.9 (dd, J = 18.3,
5.2 Hz, 1 H), 3.7 (s, 3 H), 1.8 (br s, 2 H).
13C{1H} NMR (CDCl3, 100 MHz):  = 169.1, 163.9 (t, J = 29.4 Hz), 136.0,
128.6, 128.5, 128.0, 116.0 (dd, J = 257.7, 255.8 Hz), 57.7 (dd, J = 25.0,
23.1 Hz), 52.6, 41.0.
19F NMR (CDCl3, 376 MHz):  = –111.9 (dd, J = 255.0, 10.1 Hz, 1 F),
–118.7 (dd, J = 255.0, 16.3 Hz, 1 F).
MS (EI): m/z = 272 [M]+.
Eluent: EtOAc/hexane (1:4); yield: 68.7 mg (81%); dr 1:2; colorless
solid.
1H NMR (DMSO-d6, 400 MHz):  = 9.4 (d, J = 7.3 Hz, 0.33 H), 9.3 (d, J =
7.3 Hz, 0.67 H), 7.5–7.4 (m, 2 H), 7.4–7.2 (m, 8 H), 7.0 (m, 2 H), 6.7 (m,
2 H), 6.5 (m, 1 H), 6.4 (m, 1 H), 5.5 (m, 1 H), 5.4–5.2 (m, 1 H), 3.6 (s, 1
H), 3.6 (s, 2 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 169.8, 162.6 (t, J = 28.9 Hz),
146.5, 146.3, 135.4, 135.3, 135.2, 135.0, 128.7, 128.6, 128.6, 128.3,
128.3, 128.1, 128.0, 127.9, 127.6, 127.6, 117.1, 115.8 (dd, J = 259.6,
253.8 Hz), 113.5, 113.4, 57.8 (dd, J = 27.8, 22.3 Hz), 56.2, 56.1, 52.3,
52.3.
HRMS (EI): m/z [M]+ calcd for C12H14F2N2O3: 272.0972; found:
272.0979.
Methyl (3-[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-
acetamido]-2,2-difluoro-3-phenylpropanoyl)glycinate (6)
19F NMR (DMSO-d6, 376 MHz):  = –106.6 (dd, J = 250.7, 7.9 Hz, 0.67
F), –107.0 (dd, J = 252.5, 8.1 Hz, 0.33 F), –116.3 (d, J = 252.5, 20.0 Hz,
0.37 F), –117.0 (dd, J = 250.7, 21.4 Hz, 0.67 F).
MS (EI): m/z = 424 [M]+.
HRMS (EI): m/z [M]+ calcd for C24H22F2N2O3: 424.1598 found:
To a vial containing dipeptide 5 (54 mg, 0.2 mmol), Fmoc-glycine (62
mg, 0.21 mmol), and HOBt (29.7 mg, 0.22 mmol) in DMF (1 mL) was
added EDC (42 mg, 0.22 mmol) in DMF (1.5 mL) at r.t. Then, the mix-
ture was stirred at r.t. for 22 h. The mixture was poured into aq 10%
HCl and extracted with EtOAc. The combined organic phases were
washed with aq 10% HCl, sat. aq NaHCO3 (2 ×) and brine, dried (Mg-
SO4), and concentrated in vacuo. The obtained solid was collected by
suction filtration and washed with cold Et2O to give the pure product
6 as a colorless solid; yield: 88.6 mg (80%); mp 185.5–189.0 °C.
424.1607.
Methyl [2,2-Difluoro-3-phenyl-3-(phenylamino)propanoyl]-
glycylglycylglycinate (3v)
This product was purified without flash column chromatography. The
reaction mixture was diluted with CHCl3, then the precipitated solid
was collected via suction filtration. The filtrate was washed with cold
EtOAc; yield: 75.9 mg (82%); colorless solid; mp 206.0–207.0 °C.
1H NMR (DMSO-d6, 400 MHz, 80 °C):  = 8.9 (br s, 1 H), 8.6 (br d, J =
9.6 Hz, 1 H), 7.8 (m, 2 H), 7.7 (m, 2 H), 7.4–7.2 (m, 10 H), 5.7 (td, J =
14.8, 9.8 Hz, 1 H), 4.3–4.1 (m, 3 H), 3.9–3.7 (m, 4 H), 3.6 (s, 3 H).
1H NMR (DMSO-d6, 400 MHz):  = 8.8 (t, J = 5.5 Hz, 1 H), 8.2 (t, J = 5.7
Hz, 1 H), 8.1 (t, J = 5.7 Hz, 1 H), 7.5 (m, 2 H), 7.4–7.3 (m, 3 H), 7.0 (m, 2
H), 6.7 (m, 2 H), 6.5 (m, 1 H), 6.3 (d, J = 10.5 Hz, 1 H), 5.3 (ddd, J = 20.6,
10.5, 8.7 Hz, 1 H), 3.9–3.7 (m, 6 H), 3.6 (s, 3 H).
13C{1H} NMR (DMSO-d6, 100 MHz):  = 168.9, 168.9, 162.7 (t, J = 28.9
Hz), 156.3, 143.7, 140.6, 134.1, 128.4, 128.2, 128.1, 127.5, 126.9,
125.1, 119.9, 115.3 (t, J = 257.2 Hz), 65.6, 53.7 (t, J = 24.1 Hz), 51.7,
46.5, 43.0, 40.6.
13C{1H} NMR (DMSO-d6, 100 MHz):  = 170.7, 169.6, 168.6, 163.7 (t,
J = 28.9 Hz), 147.1, 135.8, 129.4, 129.3, 128.6, 117.9, 116.6 (dd, J =
260.6, 254.3 Hz), 114.3, 58.7 (dd, J = 27.9, 22.1 Hz), 52.2, 42.6, 42.3,
41.1.
19F NMR (DMSO-d6, 376 MHz, at 80 °C):  = –111.1 (s, 2 F).
MS (FAB): m/z = 552 [M + H]+.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

I
A. Tarui et al.
Paper
Synthesis
HRMS (FAB): m/z [M + H]+ calcd for C29H28F2N3O6: 552.1946; found:
MS (EI): m/z = 291 [M]+.
552.1946.
HRMS (EI): m/z [M]+ calcd for C18H20F2N2O: 291.1071; found:
291.1074.
Kinetic Study on the Stepwise Synthesis of 3a via Alcoholysis
Intermediate (Scheme 2)
To a vial containing difluoro--lactam 2a (51.9 mg, 0.2 mmol) in the
corresponding alcoholysis solvent (0.3 mL) was added DIPEA (0.035
mL, 0.2 mmol) at r.t. and stirred at r.t. for 2 h. The full conversion of 2a
to 7 or 8 was monitored by TLC analysis. To the reaction mixture were
added glycine methyl ester hydrochloride (50.0 mg, 0.4 mmol) and
DIPEA (0.07 mL, 0.4 mmol) and the mixture was stirred at r.t. for 0.5
h. The mixture was poured into H2O and then extracted with EtOAc.
The combined organic phases were washed with brine, dried (Mg-
SO4), and concentrated in vacuo. The crude mixture was purified with
flash column chromatography to give the pure product 3a.
Funding Information
This work was supported by JSPS KAKENHI Grant Number
JP16K18855.
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Acknowledgment
We thank James Murray, Ph.D. of Edanz Group (www.edanzedit-
ing.com) for editing a draft of this manuscript.
Alcoholysis of 2a for the Structural Determination of Intermedi-
ates 7 and 8
Supporting Information
Supporting information for this article is available online at
2,2,2-Trifluoroethyl 2,2-Difluoro-3-phenyl-3-(phenylamino)-
propanoate (7)
https://doi.org/10.1055/s-0040-1707238.
S
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orit
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o
n
To a vial containing difluoro--lactam 2a (51.9 mg, 0.2 mmol) in TFE
(0.3 mL) was added DIPEA (0.035 mL, 0.2 mmol) at r.t. and the mix-
ture was stirred at r.t. for 2 h. The mixture was poured into H2O and
extracted with EtOAc. The combined organic phases were washed
with brine, dried (MgSO4), and concentrated in vacuo. The crude mix-
ture was purified by flash column chromatography (10% EtOAc in
hexane) to give the pure product 7 as a colorless solid; yield: 40.9 mg
(59%); mp 125.0–128.0 °C.
1H NMR (CDCl3, 400 MHz):  = 7.42–7.35 (m, 5 H), 7.16–7.12 (m, 2 H),
6.78–6.75 (m, 1 H), 6.66–6.64 (m, 2 H), 5.14 (ddd, J = 19.9, 10.3, 7.4
Hz, 1 H), 4.64–4.46 (m, 2 H), 4.37 (d, J = 10.1 Hz, 1 H).
13C{1H} NMR (CDCl3, 100 MHz):  = 162.3 (dd, J = 35.0, 32.5 Hz), 144.9,
133.0, 129.4, 129.1, 128.8, 128.2, 122.1 (q, J = 277.3 Hz), 119.6, 114.5,
114.4 (dd, J = 259.4, 255.9 Hz), 61.9 (q, J = 37.6 Hz,), 59.9 (dd, J = 27.2,
22.1 Hz).
19F NMR (CDCl3, 376 MHz):  = –73.5 (t, J = 7.9 Hz, 3 F), –107.8 (dd, J =
259.2, 7.4 Hz, 1 F), –119.8 (dd, J = 259.2, 19.9 Hz, 1 F).
References
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(3) (a) Abe, F.; Alvord, G.; Koyama, M.; Matsuda, A.; Talmadge, J. E.
Cancer Immunol. Immunother. 1989, 28, 29. (b) Chen, L.; Teng,
Y.; Xu, W. Curr. Med. Chem. 2011, 18, 964.
(4) Zhang, X.; Zhang, L.; Zhang, J.; Feng, J.; Yuan, Y.; Fang, H.; Xu, W.
J. Enzyme Inhib. Med. Chem. 2013, 28, 545.
(5) (a) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A.
T. J. Am. Chem. Soc. 1971, 93, 2325. (b) Stierle, A.; Strobel, G.;
Stierle, D. Science 1993, 260, 214.
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1993, 26, 160.
(7) Meanwell, N. A. J. Med. Chem. 2018, 61, 5822.
(8) For ribosome engineering, see: (a) Czekster, C. M.; Robertson,
W. E.; Walker, A. S.; Söll, D.; Schepartz, A. J. Am. Chem. Soc. 2016,
138, 5194. (b) Ward, F. R.; Watson, Z. L.; Ad, O.; Schepartz, A.;
Cate, J. H. D. Biochemistry 2019, 58, 4494.
MS (EI): m/z = 359 [M]+.
HRMS (EI): m/z [M]+ calcd for C17H14F5NO2: 359.0945; found:
359.0942.
Methyl 2,2-Difluoro-3-phenyl-3-(phenylamino)propanoate (8)
To a vial containing difluoro--lactam 2a (51.9 mg, 0.2 mmol) in
MeOH (0.3 mL) was added DIPEA (0.035 mL, 0.2 mmol) at r.t. and the
mixture was stirred at r.t. for 2 h. The mixture was poured into H2O
and extracted with EtOAc. The combined organic phases were washed
with brine, dried (MgSO4), and concentrated in vacuo. The crude mix-
ture was purified by flash column chromatography (5% EtOAc in hex-
ane) to give the pure product 8 as a colorless solid; yield: 54.4 mg
(93%); mp 120.5–121.5 °C.
1H NMR (CDCl3, 400 MHz):  = 7.41–7.32 (m, 5 H), 7.14–7.10 (m, 2 H),
6.75–6.62 (m, 3 H), 5.11 (ddd, J = 19.0, 9.6, 7.5 Hz, 1 H), 4.44 (br d, J =
9.6 Hz, 1 H), 3.81 (s, 3 H).
13C{1H} NMR (CDCl3, 100 MHz):  = 164.0 (dd, J = 33.2, 31.3 Hz), 145.3,
133.7, 129.3, 128.9, 128.7, 128.3, 119.2, 114.5 (dd, J = 258.2, 256.2
Hz), 114.3, 60.1 (dd, J = 27.0, 22.2 Hz), 53.6.
19F NMR (CDCl3, 376 MHz):  = –108.6 (dd, J = 257.9, 7.5 Hz, 1 F),
–119.2 (dd, J = 257.9, 19.0 Hz, 1 F).
(9) For enzyme engineering, see: Zhang, D.; Chen, X.; Zhang, R.;
Yao, P.; Wu, Q.; Zhu, D. ACS Catal. 2015, 5, 2220.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–J

J
A. Tarui et al.
Paper
Synthesis
(10) For examples of non-fluorinated -amino acids, see: (a) Sun, B.;
Pluta, R.; Kumagai, N.; Shibasaki, M. Org. Lett. 2018, 20, 526.
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Careful handling was needed for the isolation.
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