Facile synthesis of (s)-5,5-difluoronorleucine and its incorporation in biologically active peptides as an methionine mimetic

Article abstract of DOI:10.3987/COM-05-S(T)44

Both Boc and Fmoc protected 5,5-difluoronorleucines (F₂Nle) were easily prepared from commercially available amino acid derivatives. F₂Nle can be viewed as a mimic of methionine in which the sulfur atom is replaced by the CF₂ moiety. Two analogues of methionine-containing biologically active peptides (fMLP and Met-enkephalin) were prepared. The validity of CF₂-substituted methionine analogues was confirmed in their biological assay.

Full text of DOI:10.3987/COM-05-S(T)44

HETEROCYCLES, Vol. 67, No. 1, 2006
421
HETEROCYCLES, Vol. 67, No. 1, 2006, pp. 421 - 431. © The Japan Institute of Heterocyclic Chemistry
Received, 25th July, 2005, Accepted, 9th September, 2005, Published online, 13th September, 2005. COM-05-S(T)44
FACILE SYNTHESIS OF (S)-5,5-DIFLUORONORLEUCINE AND ITS
INCORPORATION IN BIOLOGICALLY ACTIVE PEPTIDES AS AN
METHIONINE MIMETIC
Satoshi Osada,* Takako Ishimaru, Hiroshi Kawasaki, and Hiroaki Kodama
Department of Chemistry and Applied Chemistry, Faculty of Science &
Engineering, Saga University, 1 Honjo, Saga 840-8502, Japan. E-mail:
osadas@cc.saga-u.ac.jp
Abstract – Both Boc and Fmoc protected 5,5-difluoronorleucines (F2Nle) were
easily prepared from commercially available amino acid derivatives. F2Nle can be
viewed as a mimic of methionine in which the sulfur atom is replaced by the CF2
moiety. Two analogues of methionine-containing biologically active peptides
(fMLP and Met-enkephalin) were prepared. The validity of CF2-substituted
methionine analogues was confirmed in their biological assay.
INTRODUCTION
Under physiological conditions, methionine can be oxidized to methionine sulfoxide (MetO). In the
presence of a strong oxidant, MetO is further oxidized to form methionine sulfone, MetO2 (Figure 1). The
latter process may be only of pathophysiological and/or experimental relevance, whereas the former
process is a phenomenon that can happen in living cells.
O
S O
O
S
S
CF2
H2N
COOH
H2N
COOH
H2N
COOH
H2N
COOH
Met(O)
Met(O)2
5,5-difluoronorleucine (F2Nle)
Met
Figure 1. Methionine side-chain oxidation and 5,5-difluoronorleucine as a mimetic of Met.
It is well known, partially oxidized methionine participates in age- or degenerative disease-related protein
modifications. In the case of methionine-containing biologically active peptides, oxidation on sulfur atom
results in substantial reduction in their activity. Recently, oxidation and the enzymatically-catalyzed
reduction of methionine have been in focus as a potential novel molecular mechanism for cellular

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HETEROCYCLES, Vol. 67, No. 1, 2006
regulation.1 Thus, partially oxidized methionine residues may not only degenerative modifications, but
also play a role in post-translational modification cycles of the proteins involved in cellular signaling,
which in turn may give rise to activity-dependent plastic change in cellular excitability.1b Synthetic
oxidation-resistant methionine analogues may have unique biological activity when they incorporated
into peptide, which lead to the potential to evolve into a new strategy or treating a disease, or become a
useful tool for gaining insight into the mechanisms of age-related or post-translational protein
modifications.
We designed 5,5-difluoronorleucine (F2Nle) in which the sulfur atom in methionine is replaced by a CF2
moiety as a methionine mimetic. CF2 group is often used as an isoster of oxygen,2 but its van der Waals
size is rather close to sulfur. While the sulfur atom is easily oxidized to sulfoxide in physiological
conditions, the CF2 group is inactive under such conditions. Therefore, F2Nle can be viewed as an
oxidation-resistant mimic of methionine.
Preparation of racemic3 and L-form4 of F2Nle was reported, however, both synthetic routes utilized highly
toxic and hazardous HF and SF4 to introduce fluorine. Absence of a more practical and facile synthetic
method can be one of the reasons that peptide chemists have somehow ignored the use of F2Nle as a
methionine mimic, thus we have developed a facile synthetic method to prepare enantiomerically pure
Boc- or Fmoc-protected 5,5-difluoronorleucine, starting from commercially available glutamic acid
derivative or D-serine derived Garner’s aldehyde.5 These compounds can be readily utilized for peptide
synthesis in both solution and solid-support methods. Due to the feasibility of the oxidation-resistant
F2Nle-containing peptide analogues to replace more vulnerable methionine-containing peptides,
incorporation of F2Nle to biologically active peptide was demonstrated on a chemotactic peptide, fMLP,
and an opioid peptide, Met-enkephalin.
RESULTS AND DISCUSSION
As two fluorine atoms can be easily installed by functional transformation from the corresponding methyl
ketone (1) (Scheme 1), we started to prepare 1 by utilizing two different chiral building blocks.
HOOC
COOH
O
NH2
L-Glu
F2
C
COOH
NH2
O
N
Boc
HOOC
OH
1
NH2
D-Ser
Scheme 1.

HETEROCYCLES, Vol. 67, No. 1, 2006
423
As shown in Scheme 2, the commercially available γ-methyl L-glutamate (2) was transformed to
N,O-protected ester (3) in 62% yield through 4 steps.6 Saponification of the side-chain ester by NaOH
followed by acidification gave the free acid (4). The carboxylic acid (4) was initially converted to lithium
carboxylate using LiH, treated with excess methyllithium, then quenched by addition of
chlorotrimethylsilane7 to give the methyl ketone (1) in 80% yield. Direct addition of three equivalents of
methyllithium to carboxylic acid (4) and aqueous work-up at the last step lowered the yield (maximum
55%, with tendency to form a certain amount of tert-alcohol as a byproduct). Direct addition of
methyllithium to ester (3) at low temperatures also gave ketone (1), however, chromatographic separation
of the desired ketone (1) and unreacted ester (3) was hard to accomplish because of their close Rf values.
COOH
COOMe
i) Boc2O, NaHCO3
Dioxane-water
ii) HOSu, DCC, AcOEt
COOMe
i) LiH, THF
ii) 2 equiv. MeLi, THF
NaOH
EtOH-water
99%
1
BocN
iii) NaBH4, THF-EtOH
iv) 2,2-DMP, BF3•OEt
acetone
iii) 3 equiv. TMSCl
80%.
BocN
COOH
H2N
O
O
62% (in 4 steps)
4
3
2
i) AcCl, MeOH
ii) Boc2O, Et3N, THF
iii) 2,2-DMP, BF3•OEt
acetone
O
O
HOOC
MeCOCH=PPh3
CH2Cl2
H2, 10% Pd/C
OH
1
BocN
O
NH2
D-Ser
BocN
iv) LiBH4, THF-EtOH
v) TEMPO, NaOCl, NaBr
toluene-AcOEt-H2O
O
98%
90%
5
6
51% (in 5 steps)
Scheme 2. Synthesis of methyl ketone (1) from γ-methyl glutamate or D-serine
Alternatively, treatment of the well-known Garner's aldehyde5 with Ph3P=CHCOCH3 in dichloromethane
gave α,β-unsaturated ketone as a crystalline compound in 90% yield. Subsequent hydrogenation of the
double bond was easily achieved over 10% Pd/C to give methyl ketone (1) in 98% yield. The latter route
is easier to proceed and more efficient, however, Garner's aldehyde for this scheme should be derived
from the expensive D-serine in 5 steps.
The carbonyl group of 1 was then converted to CF2 functionality in neat diethylaminosulfur trifluoride8
(DAST). The reaction rate is very slow in solution and took a week for consumption of the starting
material. Partial acetonide deprotection by trace amounts of HF was observed during the long reaction
period, which leads to a complex mixture. Complete acetonide deprotection of the difluoro compound (7)
was then achieved by treating it with Dowex resin in MeOH to give the amino alcohol (8) in 98% yield.

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HETEROCYCLES, Vol. 67, No. 1, 2006
Conversion of Boc-amino alcohol (8) to Boc-amino acid (9) was cleanly accomplished in 86% yield by
environmentally benign TEMPO-NaOCl-n-Bu4NBr oxidation.9 In contrast to the conventional PDC
oxidation in DMF, the biphasic condition of TEMPO oxidation makes it easy to isolate the amino acid by
a simple extraction technique, without using chromatographic separation. The N-protecting Boc group
was readily transformed to Fmoc protection, which is frequently used in solid-support peptide synthesis.
CF2
CF2
CF2
TEMPO, NaOCl
n-Bu4NBr
neat DAST
Dowex-50w
COOH
1
BocHN
AcOEt-H2O
81%
BocHN
BocN
OH
O
MeOH
56%
98%
7
8
9
CF2
i) 4M HCl, dioxane
COOH
FmocHN
10
ii) FmocOSu, Na2CO3
dioxane-water
81%
Scheme 3. Synthesis of N-Protected F2Nle
Thus obtained F2Nle was applied for peptide synthesis. To investigate the validity of CF2-substituted
methionine analogues, F2Nle analogues of methionine-containing biologically active peptides, fMLP and
Met-enkephalin were designed (Figure 2).
CF2
S
H
N
H
N
N
H
Leu Phe OMe
N
H
Leu Phe OH
O
O
O
O
[F2Nle1]-fMLP
fMLP
S
CF2
H–Tyr–D-Ala–Gly–Phe
H–Tyr–D-Ala–Gly–Phe
N
H
COOH
N
H
COOH
[D-Ala2]-Met-enkephalin
[D-Ala2,F2Nle5]-Met-enkephalin
Figure 2. F2Nle analogues of two biologically active peptides
As a chemotactic peptide fMLP analog, HCO-F2Nle-Leu-Phe-OMe ([F2Nle1]-fMLP) was prepared by
conventional coupling procedure between Boc-F2Nle-OH and H-Leu-Phe-OMe using EDCI and HOBt,

HETEROCYCLES, Vol. 67, No. 1, 2006
425
and successive conversion on N-terminal Boc to formyl by HCOOH and EEDQ. For a Met-enkephalin
analogue, considering ease of biological assay, [D-Ala2]-Met-enkephalin was chosen for the reference
compound, and its analog with F2Nle substitution at methionine residue was prepared by solid-support
peptide synthesis procedure using Wang resin.
Table 1. Superoxide production assay of fMLP analogues
superoxide production (EC50)
fMLP
20 nM
50 nM
[Nle1]-fMLP
[F2Nle1]-fMLP
5 nM
Table 2. Competition binding assay of Met-Enkephalin analogues
Binding Potency (IC50)
µ"OR
!-OR
#"OR
[D-Ala2]-Met-enkephalin
4 nM
4 nM
4 nM
5 nM
6200 nM
[D-Ala2,Nle5]-Met-enkephalin
[D-Ala2,F2Nle5]-Met-enkephalin
!5800 nM
5 nM
6 nM
> 10,000 nM
Biological evaluation on F2Nle analogues of methionine-containing biologically active peptides, fMLP
and Met-enkephalin, are shown in Tables 1 and 2, respectively. In both cases, Nle analogues were
independently prepared and used for evaluating feasibility of CF2 group.
Superoxide production assay were applied for fMLP analogues.10 Interestingly, superoxide production of
neutrophils stimulated by [F2Nle1]-fMLP (EC50=5 nM) was more potent than fMLP (EC50=20 nM). Some
evidences are reported that stimulated neutrophils inactivate fMLP by oxidizing the sulfur of the
methionine residue, and it has been proposed that such oxidation may be a mechanism by which
neutrophils modulate the inflammatory response.11 Slightly enhanced activity of F2Nle analog may imply
that the oxidation-resistant difluoromethylene group mimicking sulfur atom without loss of its activity.
For enkephalin analogues, binding potency of peptides for the δ, µ, and κ-opioid receptors (ORs) were
evaluated by the competition binding assay using selective antagonists and COS-7 cells expressing opioid
receptors.12 In this case, three enkephalin analogues had similar potency and selectivity trends, thus no
outstanding effect due to the CF2 substitution was observed in compared with CH2 substitution. It can be
concluded that the sulfur atom in the Met-enkephalin is not a key group for the receptor subtype

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discrimination. More detailed biological analyses for the downstream events are being undertaken, and
will be reported elsewhere.
In conclusion, we have developed a facile synthesis of 5,5-difluoronorleucine as a mimetic of methionine,
and demonstrated its sulfur atom mimicking in two biologically active peptides. Generally, Nle
substitution at Met is the first choice for increasing stability in biologically active peptides. When it
results in a decrease in activity, CF2 substitution may be one consideration for mimicking the sulfur atom
in designing stable isoster.
EXPERIMENTAL
Melting points were recorded on Yanako micro melting point apparatus MPS3 and are uncorrected. All
1
commercial reagents were used without further purification unless otherwise noted. The H-NMR,
13C-NMR and F-NMR spectra were determined on a JEOL AL-300 spectrometer (300 MHz, 75 MHz
19
and 283MHz, respectively). All chemical shifts are reported in ppm as δ value relative to internal
tetramethylsilane (1H and 13C) or benzotrifluoride (19F) in CDCl3 unless otherwise noted. Optical rotations
were recorded on a JASCO DIP-370 polarimeter. IR spectra were recorded on a JASCO IR-700
spectrophotometer as KBr disks or Nujol mulls. HRMS spectra were obtained on a JEOL GCmateII mass
spectrometer. Molecular weight of synthetic peptides was determined by MALDI-TOF MS analysis on
Voyager DERF (PerSeptive Biochem). Tetrahydrofuran was distilled from sodium benzophenone ketyl in
prior to use. All the manipulations with air-sensitive reagents were carried out under a dry argon
atmosphere. Analytical TLC was performed using Merck Silica Gel 60 F254 plates (0.25 mm on glass).
Flash column chromatography was performed using either Wakogel C-300 (45-75 µm) or Silica Gel 60
(40–63 µm). Following abbreviations are used; DIEA, N,N-diisopropylethylamine; EEDQ, N-ethoxy-
carbonyl-2-ethoxy-1,2-dihydroquinone; EDCI•HCl; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride; Fmoc, 9-fluorenylmethoxycarbonyl; Fmoc-OSu, N-(9-fluorenylmethoxycarbonyl)
succinimide; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt,
1-hydroxybenzotriazole; NMP, N-methylpiperidone; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy free
radical; TIS, triisopropylsilane.
(S, E)-tert-Butyl 2,2-dimethyl-4-(3-oxobut-1-enyl)oxazolidine-3-carboxylate (6)
To a solution of (R)-tert-butyl 4-formyl-2,2-dimethyloxazolidine-3-carboxylate (Garner’s aldehyde, 2.30
g, 10.0 mmol) in dichloromethane (60 mL) was added (acetylmethylene)triphenylphosphorane (4.00 g,
12.5 mmol) at rt with stirring. After stirring for 3 days, the solvent was evaporated in vacuo and 50 mL of
ether was added to the residue to precipitate triphenylphosphine oxide. Precipitates were filtered off, and
the filtrate was concentrated and purified by flash chromatography (hexane/ethyl acetate = 3/1) to give

HETEROCYCLES, Vol. 67, No. 1, 2006
427
title compound as colorless crystals (2.42 g, 60%). mp 76-77°C (P. ether); [α]D +62.0° (c 1.0, MeOH);
1H-NMR (mixture of Boc rotamers) δ 6.68–6.65 (m, 1H), 6.15 (t, J =15.9 Hz, 1H), 4.55–4.48 (m, 1H),
4.11 (dd, J = 6.7, 6.5 Hz, 1H), 3.80 (dd, J = 9.2, 2.0 Hz, 1H), 2.28 (s, 3H), 1.67-1.42 (m, 15H); 13C-NMR
(mixture of Boc rotamers) δ 198.1, 151.5, 144.8, 144.5, 131.2, 130.9, 94.5, 94.0, 80.7, 80.2, 67.4, 67.2,
58.2, 28.3, 27.1, 26.5, 24.5, 23.5; IR (neat film from CH2Cl2) ν 2970, 2926, 2870, 1690, 1627, 1473, 1450,
1374, 1249, 1202, 1171, 1093, 1053, 974, 852, 768 cm-1; HRMS (FAB) (M)+ calculated for C14H23NO4
269.1627, found 269.1610.
(S)-tert-Butyl 2,2-dimethyl-4-(3-oxobutyl)oxazolidine-3-carboxylate (1)
1) via ester (3).
To a solution of methyl ester (3) (3.10 g, 10.7 mmol) dissolved in 5 mL of ethanol was added NaOH
aqueous solution (0.46 g / 12 mL) at rt, then mixture was stirred for 14 h. The aqueous solution was
washed with ether, acidified by using 6M HCl aqueous solution. Free carboxylic acid was extracted with
ether, washed with brine, and dried over MgSO4. Solvent was removed in vacuo and the residue was
azeotroped with benzene to give colorless oil (2.91 g). Homogeneity of the acid was confirmed by TLC
analysis (hexane/ethyl acetate = 3/1 and CHCl3/MeOH/AcOH = 95/5/1). The carboxylic acid (4) (1.93 g)
dissolved in 20 mL of THF was added to a suspension of 90% LiH (66 mg, 7.4 mmol) in THF (10 mL) at
rt. After stirring 10 min at the ambient temperature, the mixture was cooled to 0°C, a solution of
methyllithium (1.2 M ether solution, 12 mL, 14 mmol) was added with vigorous stirring. After 1 h at 0°C,
3.0 mL (22 mmol) of chlorotrimethylsilane was added rapidly and the mixture was gradually warm up to
rt. The mixture was poured onto saturated NH4Cl aqueous solution. Phases were separated, the aqueous
layer was extracted with ether. Combined organic phases were washed with saturated NaHCO3, brine,
then dried. Solvent was removed in vacuo and flash chromatography (hexane/ethyl acetate = 3/1) gave
1.52 g of methyl ketone (1) (80%) as colorless oil.
2) via unsaturated ketone (5)
To a solution of unsaturated ketone (5) (1.33 g 4.93 mmol) in ethanol (50 mL) containing 10% Pd/C (80
mg) was stirred for 6 h at rt under hydrogen atmosphere (1 atm). Catalyst was filtered off, solvent was
removed in vacuo and flash chromatography (hexane/ethyl acetate = 3/1) gave colorless oil (1.31g, 98%).
[α]D +21.8° (c 1.0, MeOH); 1H-NMR (mixture of Boc rotamers) δ 3.86 (m, 2H), 3.62 (d, J =7.5 Hz, 1H),
2.39 (t, J = 7.6 Hz, 2H), 2.09 (s, 3H), 1.90-1.76 (m, 2H), 1.49-1.41 (m, 15H); 13C-NMR (mixture of Boc
rotamers) δ 208.0, 207.8, 152.5, 93.8, 93.3, 80.0, 79.7, 67.0, 56.5, 56.4, 40.1, 39.9, 30.8, 29.8, 28.3, 26.6,
24.3, 22.9; IR (neat) ν 2968, 2928, 2870, 1686, 1473, 1448, 1387, 1307, 1251, 1202, 1169, 1099, 1077,
1048, 1025, 853, 767 cm-1; HRMS (FAB) (M)+ C14H25NO4 271.1784, found 271.1760.

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HETEROCYCLES, Vol. 67, No. 1, 2006
(S)-tert-Butyl 4-(3,3-difluorobutyl)-2,2-dimethyloxazolidine-3-carboxylate (7)
A solution of methyl ketone (1) (1.08 g, 3.97 mmol) in diethylaminosulfur trifluoride (2 mL) was stirred
for 20 h at rt. The reaction mixture was diluted with 20 mL of ether, and the solution was carefully added
to a saturated NaHCO3 aqueous solution in dropwise. The two-phase mixture was stirred for 30 min, the
ether layer was washed with NaHCO3 aqueous solution and brine, then dried over Na2SO4. Concentration
and flash chromatography (hexane/AcOEt = 4/1) gave 647 mg (56%) of pale yellow oil. [α]D +27.9° (c
1
1.0, MeOH); H-NMR (mixture of Boc rotamers) δ 3.98-3.85 (m, 2H), 3.71 (d, J=7.6 Hz, 1H), 2.00-1.72
13
(m 4H), 1.60 (t, J=18.4 Hz, 3H), 1.56 (brs, 3H), 1.54 (s, 12H); C-NMR (mixture of Boc rotamers) δ
152.2, 151.7, 123.8 (t, J=238 Hz) 93.8, 93.3, 80.0, 79.6, 66.7, 66.5, 56.7, 56.6, 34.4 (t, J=26 Hz), 28.3,
19
27.5, 26.6, 26.3, 24.3, 23.5, 23.2, 22.9, 22.8; F-NMR δ -92 (m); IR (neat) ν 2970, 2932, 2870, 1690,
1450, 1386, 1319, 1251, 1206, 1171, 1094, 1055, 905, 852, 805, 767 cm-1; HRMS (FAB) (M+H)+
+
calculated for C14H26NO3F2 294.1875, found 294.1879.
(S)-2-(N-tert-Butyloxycarbonyl)amino-5,5-difluorohexan-1-ol (8)
To a solution of acetonide (7) (641 mg, 2.29 mmol) dissolved in 30 mL of methanol was added
Dowex-50w resin (600 mg), which was previously washed with methanol prior to use. After the mixture
was stirred for 20 h at ambient temperature, the resin was filtered and a few drop of triethylamine was
added to the filtrate. Concentration and flash chromatography (hexane/AcOEt = 2/1) gave Boc-amino
1
alcohol (8) (500 mg, 90%) as colorless oil. [α]D -15.0° (c 1.0, MeOH); H-NMR δ 4.70 (br s, 1H),
3.68-3.54 (m, 3H), 3.00 (br s, 1H), 2.00-1.81 (m, 2H), 1.80-1.04 (m, 2H), 1.60 (t, J=18.3 Hz, 3H), 1.44 (s,
13
9H); C-NMR δ 156.3, 123.9 (t, J=238 Hz) 79.9, 65.2, 52.3, 34.6 (t, J=26 Hz), 28.3, 28.3, 24.7 (t, J=4
19
Hz), 23.4 (J=28 Hz); F-NMR δ -93 (m); IR (neat) ν 3428, 3332, 2968, 2930, 2870, 1683, 1522, 1508,
+
1450, 1389, 1364, 1274, 1243, 1169, 1047, 899 cm-1; HRMS (FAB) (M+H)+ calculated for C11H22NO3F2
254.1562, found 254.1581.
N-tert-Butoxycarbonyl-5,5-difluoro-L-norleucine (9)
To a solution of Boc-amino alcohol (8) (480 mg, 1.89 mmol) dissolved in 4 mL of AcOEt and 2 mL of
water was added TEMPO (3 mg, 20 µmol), n-Bu4NBr (6 mg, 20 µmol), and NaHCO3 (600 mg, 7.14
mmol). To this biphasic mixture was added dropwise 5 mL of a solution of NaOCl (10% aqueous) with
vigorous stirring at 0°C. After the addition, the mixture was stirred for 30 min at 0°C. Phases were
separated and organic layer was extracted with saturated NaHCO3 aqueous solution. Combined aqueous
phases were treated with NaHSO3 to decompose excess NaOCl, acidified by adding 6M HCl. The
suspension was extracted with ether, and the extracts were dried over MgSO4. Solvent was removed in
vacuo, and the residue was azeotroped with benzene to remove a trace amount of water to give colorless

HETEROCYCLES, Vol. 67, No. 1, 2006
429
oil (410 mg, 81%). Compound homogeneity was confirmed by TLC analysis (hexane/ethyl acetate = 3/1
1
and CHCl3/MeOH/AcOH = 95/5/1). [α]D -11.0° (c 1.0, MeOH); H-NMR (aggregates mixture) δ 9.96
(brs, 1H), 6.95, 5.11 (2 br d, 1H), 4.36, 4.23 (2 br s, 1H), 2.19-1.81 (m, 4H), 1.60 (t, J=18.3 Hz, 3H), 1.45
1
(s, 9H); H-NMR (DMSO-d6) δ 12.54 (brs, 1H), 7.15 (d, J=8.1 Hz, 1H), 3.90 (br t, 1H), 2.00-1.58 (m,
13
4H), 1.58 (t, J=18.0 Hz, 3H), 1.45 (s, 9H); C-NMR δ 176.7, 175.7, 156.9, 155.5, 123.5 (t, J=238 Hz)
19
82.1, 80.5, 60.5, 53.7, 52.7 33.8, 28.2, 25.6, 23.5 (t, J=5 Hz), 14.1; F-NMR δ -92 (m); IR (neat film
from CH2Cl2) ν 3420, 3318, 2970, 1712, 1509, 1449, 1391, 1365, 1235, 1160, 851 cm-1; HRMS (FAB)
(M)+ calculated for C11H19NO4F2 267.1282, found 267.1275.
N-tert-Fluorenylmethoxycarbonyl-5,5-difluoro-L-norleucine (10)
Boc-F2Nle-OH (9) (133 mg, 0.5 mmol) was dissolved in 4 M HCl/dioxane and the solution was stirred for
30 min at rt. Solvent was removed in vacuo, and azeotroped with dioxane to remove excess HCl.
Obtained oil was triturated in petroleum ether-ether to give 5,5-difluoro-L-norleucine hydrochloride as a
white powder (101 mg). The powder was dissolved in 5 mL of 10% Na2CO3 aqueous solution. This
solution was added dropwise to a solution of Fmoc-OSu (235 mg) in 3 mL of dioxane. After stirring 7 h
at rt, precipitates were filtered. The filtrate was neutralized with 2 M HCl and extracted with EtOAc, the
organic extracts were washed with brine and dried over Na2SO4. Solvent was removed in vacuo, and the
resultant oil was triturated in petroleum ether to give white powder. Thus obtained compound was
immediately used for solid-phase peptide synthesis without further purification.
HCO-F2Nle-Leu-Phe-OMe (11)
To a solution of H-Leu-Phe-OMe (40 mg, 0.12 mmol) in DMF (5 mL), were added Et3N (12 mg, 0.12
mmol), Boc-F2Nle-OH (9) (33 mg, 0.12 mmol), and HOBt (18 mg, 0.13 mmol). The mixture was stirred
and cooled to 0°C, then added EDCI•HCl (25 mg, 0.13 mmol). The reaction mixture was stirred for 30
min at 0°C, and for 5 h at rt. After removing the solvent in vacuo, the residue was solidified by adding
water. Formed white solid was washed successively with 0.5 M NaHCO3, 5 % KHSO4 and water: yield
42 mg. The Boc-F2Nle-Leu-Phe-OMe (24 mg, 0.05 mmol) was treated with formic acid (5 mL) for 5 h at
rt. When TLC showed absence of starting material, formic acid was removed in vacuo, and the residue
was dissolved in 2 mL of dry THF. To the solution was added EEDQ (15 mg, 0.06 mmol) in dry THF (2
mL), and the reaction mixture was stirred for 30 min at 0°C and for 5 h at rt. After removing the solvent
in vacuo, the residue was solidified by water to give a white solid, which was successively washed with
0.5 M NaHCO3, 5 % KHSO4 and water. Yield 21 mg (91%) Peptide homogeneity was confirmed by
1
RP-HPLC. mp. 150.5-152.0°C; H-NMR (DMSO-d6) δ 8.36 (d, J=7.5 Hz, 1H), 8.29 (d, J=7.5 Hz, 1H),
8.11 (d, J=7.5 Hz, 1H), 8.01 (s, 1H), 7.29-7.18 (m, 5H), 4.50-4.30 (m, 3H), 3.57 (s, 3H), 3.06-2.11 (m,

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HETEROCYCLES, Vol. 67, No. 1, 2006
2H), 1.90-1.50 (m, 7H), 1.19 (m, 1H), 1.43-1.38 (m, 2H), 0.86 (dd, 6H). Amino acid ratios in acid
hydrolysate: Leu 1.1, Phe, 0.9. MS (ESI) (M+Na)+ m/z calculated for C23H33N3O5F2Na+ 492.23, found
492.85.
H-Tyr-D-Ala-Gly-Phe-F2Nle-OH (12)
Wang resin (L = 0.73 mmol/g, 71 mg, 0.05 mmol), which previously swelled with DMF, was placed in a
reaction vessel, and Fmoc-F2Nle (10) (54 mg, 0.15 mmol) in 1.5 mL of DMF was added and mixed 15
min. Then, pyridine (40 µL, 0.5 mmol) and 2,6-dichlorobenzoyl chloride (40 µL, 0.3 mmol) were added
and the mixture was stirred for 24 h. The resin was washed with DMF (5 times), then treated with 0.1M
DMAP/DMF (0.1 mL) and benzoic anhydride/NMP (56.5 mg/1 mL) to cap unreacted functional group.
The following steps were carried out for elongation of amino acid residues: (1) deprotection of Fmoc
group with 20% piperidine in DMF (1 min and then 10 min treatment), (2) washing with DMF (2 mL x
10), (3) coupling with activated acid esters which were prepared from the reaction of the corresponding
Fmoc-amino acids and DIEA (2.0 equiv.), 0.45M HBTU-HOBt mixture in DMF (1.0 equiv.), in 2 mL of
DMF, and (4) washing, DMF (2 mL x 10). The coupling time was for about 50 min, and usually double
coupling was applied. The completion of reaction was monitored by the Kaiser test. After coupling each
residue to the resin step by step, the desired peptide was cleaved off by using TFA/water/TIS mixture
(95/2.5/2.5, v/v/v) for 90 min. The TFA solution was concentrated and the residue was washed with ethyl
ether three times to give a white precipitate. The precipitate was dried and purified by gel permission
chromatography (Sephadex G-15, 30% acetic acid) to give desired product (34% yield). HPLC showed
the 99% of purity. Amino acid ratios in acid hydrolysate: Tyr 0.9, Ala, 1.3, Gly 1.2, Phe, 1.1. MS (ESI)
(M+H)+ m/z calculated for C29H38N5O7F2+ 606.27, found 606.11.
Biological assays of synthetic peptides: All of the assay samples of synthetic peptides were HPLC pure.
The biological activities of fMLP analogues for human neutrophils were evaluated according to a
previously reported procedure.10 Receptor binding potencies of Met-enkephalin analogues were assessed
based on the literature procedure.12
ACKNOWLEDGEMENTS
This paper is dedicated to Prof. Barry M. Trost on the occasion of his 65th birthday. The authors
appreciate Prof. Yuhei Hamasaki and Dr. Ichiro Fujita (Department of Pediatrics, Faculty of Medicine,
Saga University) for the biological assay of fMLP analogues, and Prof. Yasuyuki Shimohigashi and Ms.
Kaname Isozaki (Department of Chemistry, Faculty of graduate School of Science, Kyushu University)
for the binding assay of Met-enkephalin analogues. The authors also appreciate Dr. Jun Nishimoto and

HETEROCYCLES, Vol. 67, No. 1, 2006
431
Ms. Mihoko Sato (Instrumental Analysis Center, Saga University) for measurement of MS spectra.
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