Fluorinated β2- and β3-amino acids: Synthesis and inhibition of α-chymotrypsin

Article abstract of DOI:10.1055/s-0029-1218743

The synthesis of a series of -fluorinated β²- and β³-amino acid derivatives is described. Stereoselective fluorination at the -carbon of the β³-amino acids was achieved by deprotonation with lithium diisopropylamide followed by treatment with N-fluorobenzenesulfonimide. Fluorination of β²-amino acids employed the chiral auxiliary (4R)-4-benzyl-2-oxazolidinone. The α-fluorinated amino acids and their non-fluorinated precursors were found to competitively inhibit α-chymotrypsin. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0029-1218743

SPECIAL TOPIC
1845
Fluorinated b2- and b3-Amino Acids: Synthesis and Inhibition of
a-Chymotrypsin
S
a
ynthesis and Inhi
i
bition o
c
f
a
-Chym
o
t
trypsinoria Peddie,a,b,1 Markus Pietsch,a,1 Karen M. Bromfield,c,2 Robert N. Pike,d Peter J. Duggan,e Andrew D. Abell*a
School of Chemistry & Physics, The University of Adelaide, Adelaide, South Australia 5005, Australia
Fax +61(8)83034358; E-mail: andrew.abell@adelaide.edu.au
b
c
d
e
Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia
Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
CSIRO Molecular and Health Technologies, Bag 10, Clayton South, Victoria 3169, Australia
Received 10 March 2010
fluorine is central to the mechanism of inhibition, and it
also allows characterization of inhibitor–enzyme com-
plexes by 19F NMR analysis.28,29
Abstract: The synthesis of a series of a-fluorinated b2- and b3-ami-
no acid derivatives is described. Stereoselective fluorination at the
a-carbon of the b3-amino acids was achieved by deprotonation with
lithium diisopropylamide followed by treatment with N-fluoroben-
zenesulfonimide. Fluorination of b2-amino acids employed the
chiral auxiliary (4R)-4-benzyl-2-oxazolidinone. The a-fluorinated
amino acids and their non-fluorinated precursors were found to
competitively inhibit a-chymotrypsin.
In this paper, we report the synthesis and assay against a-
chymotrypsin of a series of a-fluoro-b3-amino acids (1–
4), an a-fluoro-b2-amino acid 9, and the non-fluorinated
analogues 5–8 and 10 (Figure 1). The study was carried
out to investigate and extend12 the effectiveness of fluori-
nated b-amino acids to act as transition-state-analogue
based inhibitors of serine proteases. We also report at-
tempts to prepare substrates of a-chymotrypsin by replac-
ing (S)-a-phenylalanine of the known substrate, N-
Key words: b-amino acids, fluorination, crystal structure, a-chy-
motrypsin, inhibitor
The replacement of hydrogen for fluorine within a biolog-
ically active compound results in significant changes to its
physical, chemical and biological properties. The small
size and high electronegativity of fluorine influences the
basicity/acidity and reactivity of neighboring groups,
while the high strength of the carbon–fluorine bond pro-
tects fluorine atoms and hence the molecule, from meta-
bolic transformations.3–8
succinyl-phenylalanyl-p-nitroanilide
(Suc-Phe-pNA)
with a b-amino acid to generate (S)-b3-(11a) and (R)-b3-
amino acids (11b; see Scheme 3). Existing studies in this
area have shown that, with few exceptions,30,31 peptide
bonds involving b3- or b2-amino acids are proteolytically
stable.14,32 However, most of structures used in these
studies32 do not incorporate the optimum amino acid resi-
due at the P1 position,33 which would be phenylalanine for
a-chymotrypsin.14,34
These structural and reactivity effects have been put to
good use. For example, fluorinated amino acids and pep-
tides are widely used as biological tracers, mechanistic
probes and enzyme inhibitors to control blood pressure,
allergies, and tumor growth (reviewed in the introduction
to ref. 3 and 5). Much research in this area has recently fo-
cused on introducing fluorine at the a-carbon of amino
acids. This is difficult for a-fluoro-a-amino acids because
of their inherent instability,9,10 however both a-fluorinated
b2- and b3-amino acids are stable structures.4,11–13 b-Pep-
tides containing these b-amino acids have been shown to
be stable to proteolysis,14 while their backbone structures
also mimic a-peptidic hairpin turns.15 In addition, some of
these b-amino acids exhibit antimicrobial activity,16 and
they are useful synthetic precursors to b-lactam
antibiotics17 and other unnatural oligopeptides.18–23
O
BocHN
OMe
O
0,1
R
PGHN
OMe
R
PG = Ac, Bz, Boc
1–4 R = F
5–8 R = H
9 R = F
10 R = H
Figure 1 Structure of the target b3- (left) and b2-amino acid deriva-
tives (right)
Synthesis of b3-Amino Acid Derivatives
We chose to prepare a-fluoro-b3-amino acids 1–4 by a
simple stereoselective fluorination of the corresponding
b3-amino acids (see Scheme 1 and Scheme 2), which are
readily prepared by Arndt–Eistert homologation of an op-
tically active a-amino acid.35–37 Thus, reaction of methyl
ester 12 with either acetyl chloride, benzoyl chloride or di-
tert-butyl dicarbonate (in the presence of a base) gave the
required starting materials 5, 6, and 7, respectively. Ste-
Fluorinated peptidomimetics provide a basis for impor-
tant protease inhibitors, e.g., trifluoromethyl ketones are
potent inhibitors of serine proteases.24–27 The constituent
SYNTHESIS 2010, No. 11, pp 1845–1859
x
x.
x
x
.
2
0
1
0
Advanced online publication: 15.04.2010
DOI: 10.1055/s-0029-1218743; Art ID: C00410SS
© Georg Thieme Verlag Stuttgart · New York

1846
V. Peddie et al.
SPECIAL TOPIC
reoselective fluorination of b3-amino acid 5 on reaction acid 13, which was itself prepared by tandem conjugate
with lithium diisopropylamide (LDA) and N-fluorobenze- addition of optically active lithium (S)-N-benzyl-N-a-
nesulfonimide (NFSI), gave an inseparable mixture of 1a methylbenzylamide to tert-butyl cinnamate, followed by
and 1b (63:37) in a combined yield of 51% after purifica- fluorination with NFSI and N-deprotection.13 This gave
tion by chromatography (Scheme 1, Table 1). Similar re- the desired syn-a-fluoro-b3-amino acid 1a after purifica-
actions of b3-amino acids 6 and 7 gave 2 and 3, tion by recrystallization (Scheme 1). However, it should
respectively, with improved diastereomeric excesses and be noted that this synthesis is less ideal in a general sense
overall yields (Table 1). In these cases, radial chromatog- since it requires a specific crotonate or cinnamate starting
raphy gave pure samples of the major diastereoisomers, material in order to access a particular amino acid.
2a and 3a, the syn-configurations of which are consistent
The a-fluoro-b3-homophenylalanine methyl esters 4a and
with data reported in the literature4 and with the X-ray
4b were prepared by separate syn fluorination of b3-ho-
crystal structure of a related compound (see Figure 2 and
later).
mophenylalanine methyl esters 8a and 8b (Scheme 2).
These starting materials were prepared by Arndt–Eistert
Since the diastereomeric mixture 1a/1b could not be sep- homologation of natural and non-natural phenylalanine,
arated by chromatography, we chose to prepare a sample respectively. Pure samples of 4a and 4b were obtained by
of 1a by acetylation and esterification of the fluorinated recrystallization from mixtures of ethyl acetate and petro-
leum ether. The syn relative configuration of 4b was con-
Table 1 Fluorination of the Lithium Enolates of 1–3 with NFSI
firmed by X-ray crystallography (Figure 2), with
assignment of the absolute configuration based on that of
the starting a-amino acid used in the synthesis.
Compound
Products
1a/1b
Yield (%)
de (%)
26
5
6
7
51
70
75
The b3-amino acid p-nitroanilides 11a and 11b were pre-
pared from 15a and 15b, respectively, as shown in
Scheme 3. In particular, the N-Boc protecting groups of
15a and 15b were removed on treatment with trifluoro-
acetic acid (TFA) and the resulting amine TFA salts were
2a/2b
66
3a/3b
56
O
O
O
O
a, b or c
OMe
HCl⋅H2N
OMe
RHN
BocHN
OMe
BocHN
OMe
12
5 R = Ac
6 R = Bz
7 R = Boc
F
a, b
8a (3S)
8b (3R)
4a (2S,3S)
4b (2R,3R)
d, e
Scheme 2 Reagents and conditions: (a) LDA, THF, –78 °C, 1 h; (b)
NFSI, THF, –78 °C, 2.5 h, 0 °C, 2 h. 4a: 73%, 90% de; 4b: 76%, 88%
de.
O
O
+
RHN
OMe
RHN
OMe
F
F
1a R = Ac
2a R = Bz
3a R = Boc
1b R = Ac
2b R = Bz
3b R = Boc
g
O
O
f
H2N
AcHN
OH
OH
F
F
14
13
Figure 2 Molecular structure and atom labeling scheme of 4b
(ORTEP). Displacement ellipsoids are shown at the 30% probability
level for non-H atoms. H atoms are depicted as small circles of arbi-
trary radii. C16H22FNO4; M = 311.35; orthorhombic; space group
P2(1)2(1)2(1); a = 5.2431(2), b = 9.1728(3), c = 33.9923(13) Å;
U = 1634.82(10) Å3; F(000) = 664; Z = 4.
Scheme 1 Reagents and conditions: (a) AcCl, Et3N, CH2Cl2, 74%;
(b) BzCl, Et3N, CH2Cl2, 85%; (c) (Boc)2O, Et3N, 1,4-dioxane–H2O
(1:1), 89%; (d) LDA, THF, –78 °C, 1 h; (e) NFSI, THF, –78 °C, 2.5 h,
0 °C, 2 h; (f) (AcO)2O, MeCN–H2O (3:1), r.t., 21 h, 77%; (g) MeOH,
concd H2SO4, 50 °C, 66 h, 57%.
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis and Inhibition of a-Chymotrypsin
1847
coupled to succinic acid mono-tert-butyl ester,38,39 in the N,N-diisopropylethylamine (DIPEA), to give 20 with
presence of N-ethyl-N¢-(3-dimethylaminopropyl)carbodi- >95% de as determined by 1H NMR, and (ii) fluorinated
imide (EDCI) and 1-hydroxy-7-azabenzotriazole (HOAt), (on treatment with LDA/NFSI) followed by a similar
to give 16a and 16b, respectively. Hydrolysis of the con- alkylation to give 19, again in >95% de. The bulky oxazo-
stituent tert-butyl esters with TFA then gave 11a and 11b. lidinone chiral auxiliaries were removed from 19 and 20
on treatment with LiOOH (formed in situ) and the result-
ing acids 21 and 22 were esterified on reaction with meth-
anol and thionyl chloride. The protected amines 9 and 10
were then prepared by a sequence involving catalytic hy-
NO2
O
15a (S)
15b (R)
drogenation, tosylation, displacement by an azido group,
and finally catalytic hydrogenation in the presence of Boc
anhydride as shown in Scheme 4. An attempt to prepare
the azide 30 directly from 26 via Mitsunobu reaction with
diphenylphosphoryl azide, triphenylphosphine and dieth-
yl azodicarboxylate (DEAD) gave rise to dehydration to
the corresponding alkene.
BocHN
N
H
a, b
16b (R)
c
NO2
O
O
Inhibition of a-Chymotrypsin
16a (S)
t-BuO
N
H
N
H
The b-amino acids 1–11 were designed to contain an aryl
group at either the b2 or b3 positions, where a-chymo-
trypsin is known to favor a phenylalanine at the analogous
P1 position in substrates and inhibitors.14,34 Inhibition of
bovine a-chymotrypsin (bCT) and human a-chymo-
trypsin (hCT) by these b-amino acids was analyzed
spectrophotometrically, with values for Ki, i.e., the disso-
ciation constant of the enzyme–inhibitor complex, given
in Tables 2– 5. An analysis of the data for bCT and b-ami-
no acids 1a/b, 2a, 3a, 5, 8a, 9, 11a, and 11b (Tables 2– 4)
revealed a reversible mode of inhibition, as characterized
by a linear steady-state turnover of the substrate. The
same behavior was found for the interaction of hCT by in-
hibitors 1a and 5 (Table 5).
O
NO2
O
O
11a (S)
11b (R)
HO
N
H
N
H
O
Scheme 3 Reagents and conditions: (a) TFA, CH2Cl2; (b) succinic
acid mono-tert-butyl ester, EDCI, HOAt, Et3N, CH2Cl2, 16a: 20%,
16b: 69%; (c) TFA, CH2Cl2, 11a: 91%, 11: 98%.
Synthesis of b2-Amino Acid Derivatives
The fluorinated b-analogues of natural a-phenylalanine
(1a/1b, 2a, and 3a) inhibited bCT with Ki values in the
low millimolar range. The non-fluorinated derivative 5
was three-fold less potent than its fluorinated counterpart
1a/1b, while 6 and 7 were devoid of activity. The en-
hanced ability of the fluorinated derivatives (1a/1b, 2a,
and 3a) to inhibit bCT may result from hydrogen bonding
between the fluorine atom and the amide protons of the
oxy-anion hole in the active site of the protease, as postu-
The non-fluorinated (R)-b2-amino acid 10 and its a-fluor-
inated analogue 9 were prepared in 37% and 8% yields,
respectively, using a methodology11,15 previously pub-
lished by us for the preparation of the enantiomer of 9 (see
Scheme 4). In brief, the oxazolidinone 17, derived from
reaction of 3-phenylpropionic acid and (4R)-4-benzyl-2-
oxazolidinone, was: (i) stereoselectively alkylated with
BnOCH2Cl, in the presence of titanium(IV) chloride and
O
O
O
O
CO2H
CO2Me
Ph
O
Ph
O
Ph
O
N
Ph
a
N
O
O
R
R
R
c
d
b
R
Ph
Ph
Ph
Ph
19 R = F
20 R = H
Ph
21 R = F
22 R = H
23 R = F
24 R = H
17 R = H
18 R = F
e
CO2Me
CO2Me
CO2Me
CO2Me
N3
TsO
HO
BocHN
g
f
h
R
R
R
R
Ph
Ph
Ph
Ph
9 R = F
10 R = H
27 R = F
28 R = H
29 R = F
30 R = H
25 R = F
26 R = H
Scheme 4 Reagents and conditions: (a) LDA, NFSI, THF, –78→0 °C, 76%, >95% de; (b) TiCl4, DIPEA, BnOCH2Cl, CH2Cl2, 0 °C, >95%
de; (c) LiOH, H2O2, THF–H2O (4:1), 0 °C; (d) SOCl2, MeOH, 0 °C to r.t.; (e) Pd/C, H2, MeOH, 95%; (f) TsCl, Et3N, DMAP, CH2Cl2, 0 °C to
r.t.; (g) NaN3, DMF, 80 °C for 29, and NaN3, DMSO, r.t. for 30; (h) Pd/C, H2, (Boc)2O, MeOH.
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

1848
V. Peddie et al.
SPECIAL TOPIC
lated for related compounds 31 and 32 (Table 3).12 Re- Table 2). The corresponding non-fluorinated derivative
placement of the N-terminal acetyl group of 1a/1b with a 10 was inactive.
larger substituent lead to three- and six-fold enhance-
ments in potency; see data for N-Boc (3a) and N-benzoyl
(2a), respectively.
C-Terminal p-nitroanilide and N-succinyl substituents
were introduced into b3-homophenylanline in order to in-
crease the water solubility44 of the potential substrates 11a
In order to best mimic natural a-phenylalanine, we re- and 11b and these derivatives were assayed against bCT.
placed the b-phenyl substituent of the N-Boc protected b3- Cleavage of the Suc-Phe-pNA analogue (a known sub-
phenylalanine methyl esters 3a and 7 with a benzyl group; strate) proceeded with a Michaelis constant of 1.52 mM
see b3-homophenylalanine analogues 4a, 4b and 8a, 8b in (Table 4), a value that is in good agreement with literature
Table 3. Significantly, 8a [derived from natural (S)-phe- data.45,46 However, both 11a and 11b were inhibitors of
nylalanine] inhibited bCT with a Ki value of 0.56 mM, bCT, with Ki values of 1.40 and 1.21 mM, respectively
while the phenyl analogue 7 was inactive. However, 8b, (Table 4). These Ki values are similar to the Ks value of
with a non-natural R-configuration at the b-position, was Suc-Phe-pNA, where the steady state parameter Km for
inactive. The corresponding derivatives with an a-fluo- the interaction of Suc-Phe-pNA with bCT has recently
rine syn to the b-benzyl group, i.e., 4a and 4b, were also been shown to be equal to the dissociation constant of the
inactive against bCT at a concentration of 0.5 mM (anal- enzyme–substrate complex, Ks.46 Therefore, the Km of
ysis of inhibition of bCT at higher concentrations of 4a Suc-Phe-pNA can be directly compared with the Ki values
and 4b was not possible due to poor solubility). Moderate of 11a and 11b.
inhibition of a-chymotrypsin has been reported12 for the
related syn-a-fluoro-b3-homophenylalanine methyl ester
31 (Ki value of 19.1 mM, Table 3) and anti-a-fluoro-b3-
homo-phenylalanine derivative 32 (Ki = 6.44 mM,
Table 3).
Reports by Seebach and colleagues14,32,47 and by Gopi et
al.,48 have shown that the insertion of a b-residue at poten-
tial cleavage sites of a protease substrate renders it pro-
teolytically stable. Our studies show that this is also the
case with an optimum P1 group in these structures, as dis-
The fluorinated b2-homophenylalanine 9 (Table 3) repre- cussed earlier. The only enzymes known to cleave short b-
sents a new class of inhibitor of a-chymotrypsin, with peptides and b-amino acid containing peptides are b-pep-
similar potency (Ki = 0.87 mM) to the most active fluori- tidyl amino-peptidases that have been isolated from
nated b3-phenylalanine methyl esters 2a and 3a (see Gram-negative bacteria.49–54 These hydrolases specifical-
ly catalyze the cleavage of N-terminal b3-homoamino
Table 2 Ki Values for the Interaction of b3-Phenylalanine Methyl
acids within oligopeptides, amides, and esters.52
Esters with bCT
Thus, the exchange of (S)-a-phenylalanine in Suc-Phe-
pNA with (S)- or (R)-b3-homophenylalanine in 11a and
11b, respectively, does not appear to influence the initial
O
O
O
binding to bCT, rather, it simply prevents subsequent hy-
drolysis. The potency of the N-succinyl-(S)-b3-homophe-
nylalanine p-nitroanilide (11a; Table 4) against bCT is
comparable to that of the N-Boc-protected methyl ester 8a
(Table 3). However, while the R-isomer 11b shows activ-
ity against bCT, the corresponding non-natural derivative
8b is inactive (Table 3). This difference may be attributed
to the presence of the p-nitroanilide group, which is re-
ported to strongly contribute to the binding of peptides to
a-chymotrypsin and other serine proteases.55,56
α
β
BzHN
OMe
BocHN
OMe
AcHN
OMe
R1 R2
R1 R2
R1 R2
2a, 6
3a, 7
1a,b, 5
Compound R1
R2
Ki (mM)a
2.84 0.80c
0.46 0.17d
0.88 0.26e
9.50 2.49c
NIg,h
Type of ligandb
1a/1b
H/F
H/F
H
competitive inhibitor
2a
3a
5
F
competitive inhibitor
F
H
NDf
The b-amino acids 1a/b, 2a, 5, 8a, 9, 11a, and 11b
(Tables 2–4) were all competitive inhibitors of bCT, as
characterized by an intersection point of the lines above
the abscissa in the Dixon plot (see Figure 3A for a repre-
sentative example) and by parallel lines in the [S]/v versus
[I] plot (see Figure 3B for a representative example). This
competitive behavior is in accordance with literature
reports12 for the b3-homophenylalanine methyl esters 31
and 32 (Table 3).
The b3-phenylalanine methyl esters 1a and 5 were also as-
sayed against hCT, with the results shown in Table 5. The
introduction of an a-fluorine into a b3-phenylalanine gives
rise to enhanced potency against this protease, as was also
observed for bCT. However, it should be noted that the
non-fluorinated compound 5 was three times more potent
H
H
H
H
competitive inhibitor
6
H
–
–
7
H
NIg,h
a Ki values were determined by plotting the data according to Dixon.40
b Modes of inhibition were determined according to Dixon40 and
Cornish-Bowden.41
c Inhibition studies were performed at 0.3 × Km and 0.75 × Km.
d Inhibition studies were performed at 0.75 × Km and 1.5 × Km.
e The Ki value was calculated according to Cheng and Prusoff,42 and
Chou43 using IC50 = 1.53 0.45 mM, [S] = 50 mM, and Km = 67 mM.
f Not determined.
g Inhibition studies were performed at 0.15 × Km and 0.75 × Km.
h No inhibition; highest inhibitor concentration analyzed: [6] = 1 mM,
[7] = 1.6 mM. These concentrations correspond to the maximum
amount of compound soluble under the described conditions.
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis and Inhibition of a-Chymotrypsin
1849
Table 3 Ki Values for the Interaction of b2-Phenylalanine Methyl Esters and b3-Homophenylalanine Methyl Esters with bCT
O
BocHN
OMe
O
O
O
R2
BocHN
OMe
AcHN
OMe
BocHN
OMe
R1 R2
R1 R2
R1 R2
4a, 8a
4b, 8b
9, 10
31, 32
Compound
R1
R2
Ki (mM)a
NIc,d
Type of ligandb
4a
4b
8a
8b
9
F
H
F
–
H
H
H
–
NIc,d
–
H
H
F
0.56 0.15c
NIc,d
competitive inhibitor
–
0.87 0.21e
NIc,d
competitive inhibitor
–
10
31
32
–
H
H
F
F
19.1f
competitive inhibitor
competitive inhibitor
H
6.44f
a Ki values were determined by plotting the data according to Dixon.40
b Modes of inhibition were determined according to Dixon40 and Cornish-Bowden.41
c Inhibition studies were performed at 0.15 × Km and 0.75 × Km.
d No inhibition; highest inhibitor concentration analyzed: [4a] = 0.5 mM, [4b] = 0.5 mM, [8b] = 0.5 mM, [10] = 0.2 mM. These concentrations
correspond to the maximum amount of compound soluble under the described conditions.
e Inhibition studies were performed at 0.75 × Km and 1.5 × Km.
f Values taken from Ohba et al.12
Table 4 Ks and Ki Values for the Interaction of N-Succinyl Amino Acid p-Nitroanilides with bCT
NO2
R1 R2
O
O
O
H
HO
HO
N
N
N
N
H
H
H
O
O
O
NO2
Suc-Phe-pNA
11a,b
Compound
R1
–
R2
–
Ksa or Kib (mM)
1.52 0.05a
1.40 0.57b
1.21 0.58b
Type of ligandc
substrate
Suc-Phe-pNA
11a
Bn
H
H
competitive inhibitor
competitive inhibitor
11b
Bn
a Km = Ks.46,
b Inhibition studies were performed at 0.75 × Km and 1.5 × Km.
c Modes of inhibition were determined according to Dixon40 and Cornish-Bowden.41
towards the human enzyme (Table 5) compared to bCT In conclusion, we have successfully synthesized a-fluoro-
(Table 2). A similar difference is apparent on comparing b3-amino acids 1–4 via stereoselective fluorination of the
the inhibitory potency of 1 against hCT and bCT. Dixon40 corresponding b3-amino acids 5–8 on treatment with LDA
and [S]/v versus [I] plots41 reveal that 1a is a competitive and NFSI. The a-fluoro-b2-amino acid 9 was prepared us-
inhibitor of hCT. In contrast, a non-competitive mode of ing a chiral auxillary, (4R)-4-benzyl-2-oxazolidinone, to
inhibition was determined for the interaction of hCT with control the stereochemistry. The fluorinated phenylala-
5.
nine analogues 1–3 and the fluorinated b2-homophenyl-
alanine 9 were found to be reversible competitive
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

1850
V. Peddie et al.
SPECIAL TOPIC
Figure 3 Dixon plot (A) and [S]/v versus [I] plot (B) for the inhibition of bCT by compound 5 in the presence of different concentrations of
the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA: full circles, [S] = 20 mM; open circles, [S] = 50 mM. Mean values and standard devia-
tions of rates from triplicate experiments in 77 mM Tris-HCl, 20 mM CaCl2, pH 7.8 with 6% v/v DMSO and 0.011 mg/mL bCT are shown.
Linear regression in (A) gave values for slopes (0.141 0.020 mM%–1 mM–1, [S] = 20 mM; 0.053 0.012 mM%–1 mM–1, [S] = 50 mM) and ver-
tical intercepts (2.12 0.03 mM%–1, [S] = 20 mM; 1.28 0.02 mM%–1, [S] = 50 mM), which were used to calculate Ki = 9.50 2.49 mM. A
competitive mode of inhibition was concluded from intersection of the lines above the abscissa in (A) and from parallel lines in (B).
Table 5 Ki Values for the Interaction of b3-Phenylalanine Methyl
formed on plastic-backed Merck-Kieselgel KG60F254 or Polygram
Sil G/UV254 plates. Flash chromatography (positive pressure of an-
hydrous nitrogen) was performed on 230–400 mesh Merck silica
gel 60. Radial chromatography was performed with a Chroma-
totron™ apparatus with silica plates (silica gel 60 PF254 with gyp-
sum) of 5, 2 and 1 mm thickness. Ultrasonication was carried out at
r.t. for 5 min using a Branson 2510 sonicator bath. IR spectra were
measured with a Shimadzu 9201PC series FTIR spectrophotometer,
a Perkin–Elmer 1600 Series FTIR spectrophotometer or a Bruker
IFS55 Equinox FTIR Spectrophotometer fitted with Specac ‘Gold-
en Gate’ diamond micro-ATR as a neat sample (1a). 1H NMR spec-
tra (300, 500, and 600 MHz) and 13C NMR spectra (75, 126, and 151
MHz) were recorded with a Varian Unity 300, Varian Inova 500 or
Varian Inova 600 spectrometer. 19F NMR spectra (282 MHz) were
obtained with a Varian Unity 300 spectrometer. NMR spectra of 1a
and 13 were measured with a Bruker DPX 300 MHz spectrometer
(1H, 300 MHz; 13C, 75 MHz; 19F, 282.4 MHz). All NMR measure-
ments were performed in either CDCl3 or CD3OD at 23 °C. Spectra
recorded in CDCl3 were referenced to TMS (dH = 0.00 ppm) and
CDCl3 (dC = 77.0 ppm); those recorded in CD3OD were referenced
Esters with hCT
O
AcHN
OMe
R
Compound R
Ki (mM)a
Type of ligandb
1a
5
F
0.88 0.40
3.60 0.83
competitive inhibitor
non-competitive inhibitor
H
a Inhibition studies were carried out at [S] = 7.2 mM (0.2 × Km) and
[S] = 14.4 mM (0.4 × Km).
b Modes of inhibition were determined according to Dixon40 and
Cornish-Bowden.41
inhibitors of bovine and human a-chymotrypsin, whereas
a-fluoro-b3-homophenylalanine derivatives of type 4
were inactive towards these proteases. Exchange of the a-
phenylalanine in the chromogenic substrate Suc-Phe-pNA
with a b-amino acid gave (S)- and (R)-b3-homophenylala-
nine (11a and 11b, respectively) that were stable inhibi-
tors, with the same binding constant as the parent
substrate. It is noteworthy that the b-amino acid-based in-
hibitors reported in this paper are unable to adopt the typ-
1
to CHD2OD (dH = 3.31 ppm) and CD3OD (dC = 49.05 ppm). H
NMR and 13C NMR signals were assigned on the basis of two-
dimensional experiments (COSY, NOESY, and HSQC). Chemical
shifts (d) are reported in ppm. Spin multiplicities are indicated by
the following symbols: s (singlet), br s (broad singlet), d (doublet),
br d (broad doublet), dd (doublet of doublet), ddd (doublet of dou-
blet of doublet), t (triplet), and m (multiplet). Electrospray ioniza-
tion (ESI) mass spectra were detected on a Micromass/Waters LCT
TOF mass spectrometer or an Agilent G1969A LC-TOF spectrom-
eter. Elemental analyses for carbon, nitrogen, hydrogen and fluorine
ical b-strand geometry normally observed on binding to a were undertaken at the University of Otago Microanalytical Labo-
protease.57
ratory. X-ray diffraction data for 4b were collected with a Bruker
Apex II diffractometer employing Mo Ka radiation (l = 0.71073 Å)
with a graphite monochromator and CCD detector at 135 K. Data
Melting points were determined with an electrothermal melting
point apparatus or a Reichert hot stage melting point apparatus and
are not corrected. Optical rotations were measured on a Perkin–
Elmer 341 polarimeter or a Polaar 2001 polarimeter, with a 1.0 dm
cell length, at 21–23 °C in HPLC-grade solvents at l = 589 nm. [a]D
values are given in deg mL g–1 dm–1; the sample concentration val-
ues are in 10 mg mL–1. Thin-layer chromatography (TLC) was per-
collection, cell refinement and data reduction were performed using
SHELXTL,58 and refined on F2 using all data by full-matrix least
squares procedures using SHELXL-97.59 Absorption corrections
and data scaling were made via redundant data using SADABS.60
All Friedal equivalents were merged due to lack of a heavy atom
and Mo Ka radiation. CCDC-766399 contains the supplementary
crystallographic data for compound 4b. It can be obtained free of
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis and Inhibition of a-Chymotrypsin
1851
charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
mixture was reduced under vacuum and the residue was partitioned
between CH2Cl2 (10 mL) and H2O (20 mL). The aqueous layer was
further extracted with CH2Cl2 (10 mL) and the combined organic
extracts were washed with saturated NaHCO3 (10 mL), then dried
over anhydrous Na2SO4, filtered and evaporated under vacuum to
give a partially crystallized oil. Recrystallization in PE gave 1a.
Crystalline bovine pancreatic a-chymotrypsin, type II, and human
pancreatic a-chymotrypsin were obtained from Sigma (Sydney,
Australia) and used without further purification. The substrates Suc-
Ala-Ala-Pro-Phe-pNA, Suc-Phe-pNA and anhydrous DMSO were
purchased from Sigma and Fluka (Sydney, Australia), respectively.
Yield: 23 mg (57%); white solid; mp 81–82 °C; [a]D23 –53.0 (c 1.00,
CDCl3).
Fluorination of b3-Amino Acids; General Procedure A
IR (diamond micro-ATR): 3376, 2959, 1758, 1657, 1519, 1223,
1097, 704 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.38–7.28 (m, 5 H, ArH), 6.28 (d,
J = 7.5 Hz, 1 H, NH), 5.53 (ddd, J = 27.6, 8.2, 3.7 Hz, 1 H, NCH),
5.33 (dd, J = 49.0, 3.7 Hz, 1 H, CHF), 3.63 (s, 3 H, OCH3), 2.04 (s,
3 H, CCH3).
13C NMR (75 MHz, CDCl3): d = 169.6, 167.5 (d, J = 24 Hz), 135.4,
129.0, 128.9, 127.9, 89.8 (d, J = 191 Hz), 54.6 (d, J = 19 Hz), 52.5,
23.4.
19F NMR (282.4 MHz, CDCl3): d = –203.3 (dd, J = 49, 27 Hz).
A solution of n-BuLi (1.6 M in hexane, 2.2 equiv) was added to a
solution of freshly distilled i-Pr2NH (2.2 equiv) in anhydrous THF
(0.5 M) at –78 °C under N2. The reaction was stirred at –78 °C for
1 h, then a solution of the amino acid (1 equiv) in anhydrous THF
(0.5 M) was added. The mixture was stirred for 1 h at –78 °C and
then NFSI (5 equiv) in anhydrous THF (1 M) was added dropwise.
The resulting yellow mixture was stirred for 2.5 h at –78 °C and for
2 h at 0 °C. Saturated NH4Cl was added and the aqueous layer was
extracted with CH2Cl2 (3 ×). The combined organic phases were
dried over MgSO4 and the solvent was removed in vacuo. PE–
EtOAc (4:1) was added to give a white precipitate that was removed
by filtration. The filtrate was concentrated in vacuo and the crude
mixture was purified as specified for each compound.
HRMS (ESI): m/z [M + H]+ calcd for C12H15FNO3: 240.1036;
found: 240.1031.
(2S,3S)- and (2R,3S)-Methyl 3-Benzamido-2-fluoro-3-phenyl-
propanoate (2a and 2b)
Esterification of Acids; General Procedure B
The carboxylic acid was suspended in ice-cold MeOH (0.1 M) and
10% (v/v) thionyl chloride was added in portions until the carboxy-
lic acid was completely soluble. The ice-cold solution of the reac-
tion mixture was stirred for 1 h, followed by a further 18 h at r.t.
before being concentrated in vacuo to give a white solid. No purifi-
cation was necessary.
Ester 6 (196 mg, 0.69 mmol) was treated with n-BuLi (0.96 mL, 1.5
mmol, 1.6 M in hexane), i-Pr2NH (0.21 mL, 1.5 mmol) and NFSI
(1.09 g, 3.5 mmol) according to General Procedure A. The crude
product was a 83:17 mixture of 2a and 2b (determined by 19F NMR)
and was purified by radial chromatography (silica; 5 mm plate;
EtOAc–CH2Cl2, 0:1→1:9) to give a fraction containing a mixture of
2a and 2b (112 mg, 54%) and a pure fraction of 2a (34 mg, 16%).
Both fractions were off-white solids and gave a total yield of 70%.
(2S,3S)- and (2R,3S)-Methyl 3-Acetamido-2-fluoro-3-phenyl-
propanoate (1a and 1b)
Method A: Ester 5 (200 mg, 0.90 mmol) was treated with n-BuLi
(1.24 mL, 2.0 mmol, 1.6 M in hexane), i-Pr2NH (0.28 mL, 2.0
mmol) and NFSI (1.43 g, 4.5 mmol) according to General Proce-
dure A. The crude product was purified by radial chromatography
(silica; 5 mm plate; EtOAc–CH2Cl2, 0:1→1:9) to give a 63:37 mix-
ture (determined by 19F NMR) of 1a and 1b (111 mg, 51%) as a col-
orless oil.
Data for 2a (isolated):
Mp 133–135 °C (Lit.4 138–140 °C); [a]D22 –3.3 (c 1.14, CHCl3).
IR (KBr): 3412, 1764, 1713, 1521, 1456, 1289, 1231, 1107, 1048,
700 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.84–7.78 (m, 2 H, ArH), 7.58–
7.33 (m, 8 H, ArH), 6.86 (d, J = 8.0 Hz, 1 H, NH), 5.75 (ddd,
J = 26.7, 8.0, 3.7 Hz, 1 H, NCH), 5.47 (dd, J = 48.8, 3.6 Hz, 1 H,
CHF), 3.67 (s, 3 H, OCH3).
13C NMR (75 MHz, CDCl3): d = 167.4 (d, J = 23.6 Hz), 166.8,
135.2, 133.7, 132.0, 129.0, 128.8, 128.7, 127.8, 127.1, 89.6 (d,
J = 191.4 Hz), 54.9 (d, J = 18.6 Hz), 52.5.
19F NMR (282 MHz, CDCl3): d = –202.45 (dd, J = 48.8, 26.8 Hz).
Data from mixture of 1a and 1b:
IR (KBr): 3289, 3065, 2957, 1763, 1659, 1536, 1295, 1222, 1101,
732, 711 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.54 (d, J = 8.3 Hz, 1 H, NH, ma-
jor), 7.47 (d, J = 9.3 Hz, 1 H, NH, minor), 7.38–7.25 (m, 10 H,
ArH), 5.64 (ddd, J = 28.9, 9.3, 2.1 Hz, 1 H, NCH, minor), 5.51
(ddd, J = 28.1, 8.3, 3.7 Hz, 1 H, NCH, major), 5.21 (dd, J = 48.9,
3.7 Hz, 1 H, CHF, major), 5.16 (dd, J = 47.4, 2.3 Hz, 1 H, CHF, mi-
nor), 3.72 (s, 3 H, OCH3, minor), 3.59 (s, 3 H, OCH3, major), 2.02
(s, 3 H, CCH3, minor), 2.00 (s, 3 H, CCH3, major).
HRMS (ESI): m/z [M + H]+ calcd for C17H17NO3F: 302.1192;
found: 302.1180.
Selected data for minor diastereoisomer 2b from mixture:
1H NMR (300 MHz, CDCl3): d = 6.95 (d, J = 10.2 Hz, 1 H, NH),
5.94–5.87 (m, 1 H, NCH), 5.32 (dd, J = 47.4, 1.8 Hz, 1 H, CHF),
3.82 (s, 3 H, OCH3).
13C NMR (75 MHz, CDCl3): d = 168.0 (d, J = 25.1 Hz), 166.8, 90.0
(d, J = 191.0 Hz), 54.0 (d, J = 19.3 Hz), 52.7.
13C NMR (126 MHz, CDCl3): d = 170.1 (major + minor), 167.7 (d,
J = 25.3 Hz, minor), 167.2 (d, J = 23.7 Hz, major), 136.8, 135.0,
128.4, 128.3, 128.1, 127.8, 127.6, 126.6, 90.0 (d, J = 190.3 Hz, mi-
nor), 89.4 (d, J = 191.4 Hz, major), 54.1 (d, J = 18.9 Hz, major),
53.5 (d, J = 19.0 Hz, minor), 52.3 (minor), 52.0 (major), 22.4 (ma-
jor), 22.3 (minor).
19F NMR (282 MHz, CDCl3): d = –203.0 (dd, J = 47.4, 29.1 Hz, mi-
nor), –203.1 (dd, J = 48.9, 28.1 Hz, major).
HRMS (ESI): m/z [M + H]+ calcd for C12H15NO3F: 240.1036;
19F NMR (282 MHz, CDCl3): d = –202.8 (dd, J = 47.2, 27.8 Hz).
(2S,3S)- and (2R,3S)-Methyl 3-(tert-Butoxycarbonylamino)-2-
fluoro-3-phenylpropanoate (3a and 3b)
Ester 7 (214 mg, 0.77 mmol) was treated with n-BuLi (1.06 mL, 1.7
mmol, 1.6 M in hexane), i-Pr2NH (0.24 mL, 1.7 mmol) and NFSI
(1.20 g, 3.8 mmol) according to General Procedure A. The crude
product was a 78:22 mixture of 3a and 3b (determined by 19F NMR)
and was purified by radial chromatography (silica; 5 mm plate;
EtOAc–CH2Cl2, 0:1→1:9) to give a fraction containing a mixture of
found: 240.1027.
Method B: To a solution of (2S,3S)-3-acetamido-2-fluoro-3-phenyl-
propanoic acid (14; 38 mg, 0.17 mmol) in anhydrous MeOH (2 mL)
was added concentrated H2SO4 (1 drop). The reaction was stirred
under a calcium chloride drying tube at 50 °C for 66 h. The reaction
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

1852
V. Peddie et al.
SPECIAL TOPIC
3a and 3b (157 mg, 68%) and a pure fraction of 3a (13 mg, 6%).
Both fractions were off-white solids and gave a total yield of 75%.
crystallization (EtOAc–PE) gave the single stereoisomer 4b as
white needles.
Data for 3a (isolated):
Mp 88–89 °C; [a]D22 –10.0 (c 1.05, CHCl3).
Mp 82–84 °C; [a]D22 +32.4 (c 0.89, CHCl3).
IR (KBr): 3380, 1736, 1682, 1515, 1273, 1161, 1048, 1010, 740
cm–1.
1H NMR (500 MHz, CDCl3): d = 7.32–7.20 (m, 5 H, ArH), 5.04
(dd, J = 48.4, 2.6 Hz, 1 H, CHF), 4.73 (d, J = 7.7 Hz, 1 H, NH),
4.52–4.40 (m, 1 H, NCH), 3.65 (s, 3 H, OCH3), 2.88–2.82 (m, 2 H,
CH2Ph), 1.38 [s, 9 H, C(CH3)3].
13C NMR (126 MHz, CDCl3): d = 168.0 (d, J = 23.8 Hz), 154.9,
136.2, 129.4, 128.5, 126.9, 89.6 (d, J = 189.1 Hz), 80.1, 52.8 (d,
J = 21.0 Hz), 52.4, 35.2, 28.2.
19F NMR (23 °C, 282 MHz, CDCl3): d = –203.4, –203.8 (m, minor
rotamer), –204.7 (dd, J = 48.5, 24.8 Hz, major rotamer).
IR (KBr): 3389, 2978, 1767, 1712, 1497, 1368, 1293, 1250, 1167,
702 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.38–7.27 (m, 5 H, ArH), 5.43–
5.13 (m, 3 H, NH, CHF, NCH), 3.65 (s, 3 H, OCH3), 1.44 [s, 9 H,
C(CH3)3].
13C NMR (75 MHz, CDCl3): d = 167.4 (d, J = 23.5 Hz), 154.7,
135.6, 128.8, 128.6, 127.6, 90.0 (d, J = 192.0 Hz), 80.4, 55.9 (d,
J = 18.0 Hz), 52.3, 28.3.
19F NMR (282 MHz, CDCl3): d = –203.5 (dd, J = 49.1, 26.4 Hz).
HRMS (ESI): m/z [M + H]+ calcd for C15H21NO4F: 298.1455;
found: 298.1443.
HRMS (ESI): m/z [M + Na]+ calcd for C16H22NO4FNa: 334.1431;
found: 334.1425.
Data for the minor diastereoisomer 3b from mixture:
Anal. Calcd for C16H22NO4F: C, 61.72; H, 7.12; N, 4.50; F, 6.10.
Found: C, 61.58; H, 7.39; N, 4.48; F, 5.90.
1H NMR (300 MHz, CDCl3): d = 7.52–7.17 (m, 5 H, ArH), 5.49–
5.05 (m, 3 H, NH, CHF, NCH), 3.81 (s, 3 H, OCH3), 1.43 [s, 9 H,
C(CH3)3].
(R)-Methyl 3-Acetamido-3-phenylpropanoate (5)
Et3N (0.49 mL, 3.5 mmol) was added dropwise to a solution of 1267
(302 mg, 1.4 mmol) in anhydrous CH2Cl2 (5 mL) under N2 and
cooled to 0 °C. Acetyl chloride (150 mL, 1.7 mmol) was added drop-
wise and the resulting solution was stirred at 0 °C for 1 h. The reac-
tion was quenched by addition of H2O (5 mL) and the organic phase
was separated, dried (MgSO4) and concentrated in vacuo. The crude
product was purified by radial chromatography (silica; 5 mm plate;
EtOAc–PE, 4:1) to give 5 (228 mg, 74%) as a white solid. Spectral
properties are in agreement with those in the literature.61
13C NMR (75 MHz, CDCl3): d = 167.9 (d, J = 25.1 Hz), 154.8,
137.4, 128.7, 128.1, 126.6, 90.4 (d, J = 190.8 Hz), 80.2, 55.3 (d,
J = 18.3 Hz), 52.6, 28.1.
19F NMR (282 MHz, CDCl3): d = –204.0 (dd, J = 47.3, 27.8 Hz).
(2S,3S)-Methyl 3-tert-Butoxycarbonylamino-2-fluoro-4-phe-
nylbutanoate (4a)
Amino acid 8a (66 mg, 0.23 mmol) was treated with n-BuLi (0.31
mL, 0.50 mmol, 1.6 M in hexane), i-Pr2NH (70 mL, 0.50 mmol) and
NFSI (425 mg, 1.3 mmol) according to General Procedure A. The
crude product was purified by radial chromatography (silica; 5 mm
plate; EtOAc–PE, 1:9) to give 4a (51 mg, 73%) as a white solid with
a diastereomeric ratio of 95:5 (determined by 19F NMR). Recrystal-
lization (EtOAc–PE) gave the single diasteroisomer 4a as white
needles.
Mp 94–95 °C; [a]D22 +60.1 (c 0.90, CHCl3).
1H NMR (500 MHz, CDCl3): d = 7.36–7.25 (m, 5 H, ArH), 6.53 (d,
J = 6.2 Hz, 1 H, NH), 5.46–5.41 (m, 1 H, NCH), 3.62 (s, 3 H,
OCH3), 2.95 (dd, J = 15.8, 5.8 Hz, 1 H, CHH), 2.85 (dd, J = 15.8,
6.0 Hz, 1 H, CHH), 1.58 (s, 3 H, CCH3).
13C NMR (75 MHz, CDCl3): d = 171.8, 169.3, 140.4, 128.7, 127.7,
126.2, 51.8, 49.4, 39.6, 23.5.
Mp 86–87 °C; [a]D22 +13.1 (c 0.82, CHCl3).
HRMS (ESI): m/z [M + H]+ calcd for C12H16NO3: 222.1130; found:
222.1123.
IR (KBr): 3374, 1736, 1682, 1515, 1273, 1161, 1048, 1010, 740
cm–1.
1H NMR (500 MHz, CDCl3): d = 7.32–7.19 (m, 5 H, ArH), 5.03
(dd, J = 48.5, 3.0 Hz, 1 H, CHF), 4.73 (d, J = 8.0 Hz, 1 H, NH),
4.52–4.41 (m, 1 H, NCH), 3.65 (s, 3 H, OCH3), 2.90–2.81 (m, 2 H,
CH2Ph), 1.38 [s, 9 H, C(CH3)3].
13C NMR (126 MHz, CDCl3): d = 167.9 (d, J = 23.8 Hz), 154.9,
136.1, 129.4, 128.5, 126.8, 89.6 (d, J = 188.9 Hz), 80.0, 52.8 (d,
J = 20.2 Hz), 52.4, 35.2, 28.2.
19F NMR (282 MHz, CDCl3): d = –203.4, –203.9 (m, minor rotam-
er), –204.7 (dd, J = 48.4, 24.9 Hz, major rotamer).
HRMS (ESI): m/z [M + H]+ calcd for C16H22NO4FNa: 334.1430;
(R)-Methyl 3-Benzamido-3-phenylpropanoate (6)
Et3N (0.39 mL, 2.8 mmol) was added dropwise to a solution of 1267
(201 mg, 0.93 mmol) in anhydrous CH2Cl2 (8 mL) under N2 and
cooled to 0 °C. Benzoyl chloride (220 mL, 1.9 mmol) was added
dropwise and the resulting solution was stirred at 0 °C for 1 h and at
r.t. for 18 h. The reaction was quenched by addition of 1 M aqueous
HCl. The organic phase was separated and washed with sat. aq
NaHCO3 (10 mL) and NaCl (10 mL), dried over MgSO4 and con-
centrated in vacuo. The crude product was purified by radial chro-
matography (silica; 5 mm plate; EtOAc–CH2Cl2, 1:9) to give 6 (225
mg, 85%) as a white solid. Spectral properties are in agreement with
those in the literature.62
found: 334.1425.
Anal. Calcd for C16H22NO4F: C, 61.72; H, 7.12; N, 4.50; F, 6.10.
Found: C, 61.61; H, 7.07; N, 4.44; F, 6.01.
Mp 117–119 °C; [a]D22 –23.9 (c 0.98, CHCl3).
1H NMR (500 MHz, CDCl3): d = 7.83 (d, J = 7.1 Hz, 2 H, ArH, o-
ArH, Bz), 7.62 (d, J = 8.2 Hz, 1 H, NH), 7.49 (t, J = 7.4 Hz, 1 H,
ArH, p-ArH, Bz), 7.42 (t, J = 7.6 Hz, 2 H, ArH, m-ArH, Bz), 7.37–
7.24 (m, 5 H, ArH, PhH), 5.66–5.61 (m, 1 H, NCH), 3.63 (s, 3 H,
OCH3), 3.04 (dd, J = 15.8, 5.7 Hz, 1 H, CHH), 2.94 (dd, J = 15.8,
5.8 Hz, 1 H, CHH).
13C NMR (75 MHz, CDCl3): d = 172.0, 166.5, 140.5, 134.1, 131.5,
128.7, 128.5, 127.6, 127.0, 126.2, 51.8, 49.7, 39.6.
HRMS (ESI): m/z [M + H]+ calcd for C17H18NO3: 284.1287; found:
(2R,3R)-Methyl 3-(tert-Butoxycarbonylamino)-2-fluoro-4-phe-
nylbutanoate (4b)
Amino acid 8b (520 mg, 1.8 mmol) was treated with n-BuLi (2.44
mL, 3.9 mmol, 1.6 M in hexane), i-Pr2NH (0.54 mL, 3.9 mmol) and
NFSI (2.80 g, 8.9 mmol) according to General Procedure A. The
crude product was purified by radial chromatography (silica; 5 mm
plate; EtOAc–PE, 1:9) to give 4b (419 mg, 76%) as a white solid
with a diastereomeric ratio of 96:4 (determined by 19F NMR). Re-
284.1292.
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis and Inhibition of a-Chymotrypsin
1853
(R)-Methyl 3-(tert-Butoxycarbonylamino)-3-phenylpropanoate
(7)
HRMS (ESI): m/z [M + H]+ calcd for C16H24NO4: 294.1705; found:
294.1716.
b-Amino acid derivative 1267 (250 mg, 1.2 mmol) was dissolved in
a 1:1 biphasic mixture of H2O and 1,4-dioxane (7 mL). To this was
added Et3N (0.46 mL, 3.3 mmol) and di-tert-butyl dicarbonate (304
mg, 1.4 mmol). The reaction mixture was stirred at r.t. for 18 h then
concentrated in vacuo. The residue was partitioned between EtOAc
(10 mL) and 10% (w/w) aqueous citric acid (10 mL). The organic
phase was separated and the aqueous phase was extracted with
EtOAc (2 × 10 mL). The combined organic phases were washed
with sat. aq NaCl (30 mL), dried over MgSO4, filtered and concen-
trated in vacuo. The crude product was purified by radial chroma-
tography (silica; 5 mm plate; EtOAc–PE, 1:4) to give 7 (289 mg,
89%) as a white solid. Spectral properties are in agreement with
those in the literature.63
Mp 88–90 °C; [a]D22 +36.0 (c 0.98, CHCl3).
1H NMR (300 MHz, CDCl3): d = 7.38–7.25 (m, 5 H, ArH), 5.45 (br
s, 1 H, NH), 5.18–5.02 (m, 1 H, NCH), 3.62 (s, 3 H, OCH3), 2.90–
2.81 (m, 2 H, CHCH2), 1.42 [s, 9 H, C(CH3)3].
13C NMR (75 MHz, CDCl3): d = 171.4, 155.0, 141.2, 128.6, 127.5,
126.1, 79.7, 51.8, 51.2, 40.8, 28.3.
(R)-Methyl 2-Benzyl-3-(tert-butoxycarbonylamino)-2-fluoro-
propanoate (9)
A catalytic amount of Pd/C and (Boc)2O (306 mg, 1.4 mmol) were
added to a solution of 29 (276 mg, 1.2 mmol) in anhydrous MeOH
(25 mL) at r.t. The apparatus was evacuated and flushed three times
with H2 and stirred at r.t. for 18 h under an H2 atmosphere. The Pd/
C catalyst was removed by filtration through Celite and the filtrate
was concentrated in vacuo to give an oil that was purified by radial
chromatography (silica; 5 mm plate; EtOAc–PE, 3:17) to give 9
(273 mg, 76%) as a white solid.
Mp 41–42 °C; [a]D22 –24.7 (c 0.91, CHCl3).
IR (KBr): 3392, 2979, 1763, 1717, 1516, 1367, 1250, 1169, 701
cm–1.
1H NMR (500 MHz, CDCl3): d = 7.36–7.07 (m, 5 H, ArH), 4.85 (br
s, 1 H, NH), 3.86–3.74 (m, 1 H, CHHN), 3.69 (s, 3 H, OCH3), 3.52–
3.43 (m, 1 H, CHHN), 3.30–3.06 (m, 2 H, PhCH2), 1.44 [s, 3 H,
C(CH3)3].
13C NMR (151 MHz, CDCl3): d = 169.7 (d, J = 26.4 Hz), 155.6,
133.6, 130.1, 128.4, 127.3, 97.0 (d, J = 190.4 Hz), 79.9, 52.5, 45.9
(d, J = 21.9 Hz), 40.5 (d, J = 22.0 Hz), 28.2.
HRMS (ESI): m/z [M + H]+ calcd for C15H22NO4: 280.1549; found:
280.1557.
19F NMR (282 MHz, CDCl3): d = –167.8, –168.2 (m).
HRMS (ESI): m/z [M + H]+ calcd for C16H23NO4F: 312.1611;
Boc-(S)-b3-hPhe Methyl Ester (8a)
To a solution of Boc-(S)-b3-hPhe (500 mg, 1.8 mmol) in anhydrous
DMF (4 mL) under N2, were added KHCO3 (359 mg, 3.6 mmol) and
MeI (165 mL, 2.7 mmol), and the mixture was stirred at r.t. for 12 h.
The reaction mixture was diluted with EtOAc (10 mL) and the or-
ganic phase was washed with sat. aq NaCl (10 mL), dried (MgSO4)
and concentrated in vacuo. The crude product was purified by radial
chromatography (silica; 5 mm plate; EtOAc–PE, 3:17) to give 8a
(526 mg, quant) as a white solid. Spectral and physical properties
are in agreement with those reported in the literature.64
found: 312.1623.
Anal. Calcd for C16H23NO4F: C, 61.72; H, 7.12; N, 4.50. Found: C,
61.56; H, 7.14; N, 4.44.
(S)-Methyl 2-Benzyl-3-(tert-butoxycarbonylamino)propanoate
(10)
A catalytic amount of Pd/C and (Boc)2O (515 mg, 2.4 mmol) were
added to a solution of azide 30 (430 mg, 2.0 mmol) in anhydrous
MeOH (15 mL) at r.t. The apparatus was evacuated and flushed
three times with H2 and the reaction mixture was stirred at r.t. for 18
h under an H2 atmosphere. The Pd/C catalyst was removed by filtra-
tion through Celite. The filtrate was concentrated in vacuo and the
resulting oil was purified by radial chromatography (silica; 5 mm
plate; EtOAc–PE, 1:9→1:4) to give 10 (492 mg, 86%) as a colorless
oil.
Mp 48–50 °C; [a]D22 –12.3 (c 0.94, CHCl3).
1H NMR (500 MHz, CDCl3): = 7.32–7.15 (m, 5 H, ArH), 5.04 (br
d, J = 6.9 Hz, 1 H, NH), 4.21–4.13 (m, 1 H, NCH), 3.69 (s, 3 H,
OCH3), 2.97–2.78 (m, 2 H, CH2CO2CH3), 2.52 (dd, J = 15.9, 5.5
Hz, 1 H, CHHPh), 2.44 (dd, J = 15.9, 5.8 Hz, 1 H, CHHPh), 1.41 [s,
9 H, C(CH3)3].
HRMS (ESI): m/z [M + H]+ calcd for C16H24NO4: 294.1705; found:
294.1710.
[a]D22 –1 (c 1.09, CHCl3) {Lit.66 for the R-isomer [a]D +0.9 (c 1.11,
CHCl3)}.
Anal. Calcd for C16H24NO4: C, 65.51; H, 7.90; N, 4.77. Found: C,
65.24; H, 7.86; N, 4.81.
IR (KBr): 3379, 2978, 1720, 1512, 1450, 1366, 1250, 1165 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.30–7.13 (m, 5 H, ArH), 5.01 (br
s, 1 H, NH), 3.61 (s, 3 H, OCH3), 3.41–3.21 (m, 2 H, NHCH2),
2.97–2.89 (m, 2 H, PhCHHCH, PhCH2CH), 2.85–2.76 (m, 1 H, Ph-
CHHCH), 1.42 [s, 9 H, C(CH3)3].
13C NMR (126 MHz, CDCl3): d = 174.6, 155.7, 138.2, 128.8, 128.5,
126.5, 79.3, 51.7, 47.3, 41.5, 35.8, 28.3.
Boc-(R)-b3-hPhe Methyl Ester (8b)
To a solution of Boc-(R)-b3-hPhe (125 mg, 0.45 mmol) in anhy-
drous DMF (2 mL) under N2, were added KHCO3 (90 mg, 0.90
mmol) and MeI (41 mL, 0.67 mmol), and the mixture was stirred at
r.t. for 12 h. The reaction mixture was diluted with EtOAc (5 mL)
and the organic phase was washed with sat. aq NaCl (5 mL), dried
(MgSO4) and concentrated in vacuo. The crude product was puri-
fied by radial chromatography (silica; 5 mm plate; EtOAc–PE,
3:17) to give 8b (132 mg, quant) as a white solid. Spectral and phys-
ical properties are in agreement with those reported in the litera-
ture.65
HRMS (ESI): m/z [M + H]+ calcd for C16H24NO4: 294.1705; found:
294.1702.
Suc-(S)-b3-hPhe p-Nitrophenyl Ester (11a)
tert-Butyl ester 16a (33 mg, 73 mmol) was dissolved in 10% TFA in
CH2Cl2 (2 mL). The reaction mixture was stirred at r.t. for 18 h, then
concentrated in vacuo. The product was sonicated in Et2O (1 mL)
and filtered to give 11a (27 mg, 91%) as an off-white solid.
Mp 47–48 °C; [a]D22 +11.8 (c 0.25, CHCl3).
1H NMR (500 MHz, CDCl3): d = 7.32–7.17 (m, 5 H, ArH), 5.07 (br
d, J = 6.3 Hz, 1 H, NH), 4.22–4.10 (m, 1 H, NCH), 3.68 (s, 3 H,
OCH3), 2.97–2.77 (m, 2 H, CH2CO2CH3), 2.51 (dd, J = 15.8, 5.4
Hz, 1 H, CHHPh), 2.44 (dd, J = 15.9, 5.8 Hz, 1 H, CHHPh), 1.40 [s,
9 H, C(CH3)3].
Mp 155 °C (dec.).
IR (KBr): 1646, 1553, 1506, 1407, 1333 cm–1.
1H NMR (300 MHz, CD3OD): d = 8.14 (d, J = 9.1 Hz, 2 H, ArH, p-
NA), 7.74 (d, J = 9.1 Hz, 2 H, ArH, p-NA), 7.27–7.11 (m, 5 H,
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

1854
V. Peddie et al.
SPECIAL TOPIC
ArH, PhH), 4.53–4.42 (m, 1 H, NHCH), 2.95–2.76 (m, 2 H,
washings ran clear, to give 15a (151 mg, 42%) as a light-yellow sol-
CH2Ph), 2.65–2.30 [m, 6 H, CHCH2CO, (CH2)2].
id.
13C NMR (151 MHz, CD3OD): d = 176.5, 174.2, 171.9, 146.1,
144.6, 139.5, 130.5, 129.5, 127.6, 125.7, 120.5, 49.8, 42.5, 41.2,
31.9, 30.6.
HRMS (ESI): m/z [M + H]+ calcd for C20H22N3O6: 400.1509; found:
400.1525.
Mp 190–191 °C (dec.).
IR (KBr): 1682, 1504, 1342 cm–1.
1H NMR (500 MHz, CD3OD): d = 8.20 (d, J = 9.2 Hz, 2 H, ArH, p-
NA), 7.79 (d, J = 9.1 Hz, 2 H, ArH, p-NA), 7.31–7.16 (m, 5 H,
ArH, PhH), 4.27–4.19 (m, 1 H, NHCH), 2.91–2.81 (m, 2 H,
CH2Ph), 2.61 (dd, J = 14.7, 5.8 Hz, 1 H, CHHCO), 2.54 (dd,
J = 14.7, 7.8 Hz, 1 H, CHHCO), 1.33 [s, 9 H, C(CH3)3].
13C NMR (75 MHz, CD3OD): d = 172.2, 157.7, 146.2, 144.6, 139.7,
130.5, 129.4, 127.5, 125.7, 120.4, 80.1, 51.1, 43.1, 41.9, 28.7.
HRMS (ESI): m/z [M + H]+ calcd for C21H26N3O5: 400.1872; found:
Suc-(R)-b3-hPhe p-Nitrophenyl Ester (11b)
tert-Butyl ester 16b (115 mg, 0.25 mmol) was dissolved in 10%
TFA in CH2Cl2 (5 mL). The reaction mixture was stirred at r.t. for
18 h, then concentrated in vacuo. The product was sonicated in Et2O
(2 mL) and filtered to give 11b (99 mg, 98%) as an off-white solid.
400.1867.
Mp 158 °C (dec.).
IR (KBr): 1638, 1501, 1443, 1399, 1339 cm–1.
Boc-(R)-b3-hPhe p-Nitrophenyl Ester (15b)
1H NMR (500 MHz, CD3OD): d = 8.19 (d, J = 9.3 Hz, 2 H, ArH, p-
NA), 7.79 (d, J = 9.2 Hz, 2 H, ArH, p-NA), 7.30–7.16 (m, 5 H,
ArH, PhH), 4.56–4.49 (m, 1 H, NHCH), 2.94 (dd, J = 13.7, 6.3 Hz,
1 H, CHHPh), 2.87 (dd, J = 13.7, 7.9 Hz, 1 H, CHHPh), 2.66 (dd,
J = 14.9, 5.9 Hz, 1 H, CHCHHCO), 2.58 (dd, J = 14.9, 7.3 Hz, 1 H,
CHCHHCO), 2.52–2.46 (m, 2 H, CH2CH2), 2.41–2.34 (m, 2 H,
CH2CH2).
13C NMR (75 MHz, CD3OD): d = 176.6, 174.2, 171.9, 146.1, 144.5,
139.5, 130.5, 129.5, 127.6, 125.7, 120.5, 49.7, 42.4, 41.2, 31.8,
30.6.
Boc-(R)-b3-hPhe (256 mg, 0.92 mmol), p-nitroaniline (664 mg, 4.6
mmol), HATU (696 mg, 1.8 mmol), and HOAt (249 mg, 1.8 mmol)
were dissolved in anhydrous CH2Cl2 (2 mL) under N2. Et3N (0.51
mL, 3.7 mmol) was added and the reaction mixture was stirred at r.t.
for 18 h. The reaction mixture was partitioned between EtOAc (5
mL) and aq HCl (0.5 M, 5 mL). The organic phase was separated
and washed sequentially with aq HCl (0.5 M, 5 mL), and sat. aq
NaCl (5 mL). The organic phase was dried over MgSO4, filtered and
concentrated in vacuo. The crude product was recrystallized from
isopropanol, filtered and washed with ice-cold isopropanol until the
washings ran clear, to give 15b (223 mg, 61%) as a cream solid.
HRMS (ESI): m/z [M + H]+ calcd for C20H22N3O6: 400.1509; found:
400.1509.
Mp 192–193 °C (dec.).
IR (KBr): 1683, 1506, 1336 cm–1.
(2S,3S)-3-Acetamido-2-fluoro-3-phenylpropanoic Acid (14)
To a solution of (2S,3S)-3-amino-2-fluoro-3-phenylpropanoic
acid13 (13; 57 mg, 0.31 mmol) in MeCN–H2O (3:1, 4 mL) was add-
ed gradually over 3 h, Ac2O (180 mL, 1.86 mmol). The reaction was
stirred for 18 h then the reaction mixture was diluted with H2O (20
mL) and extracted with EtOAc (2 × 20 mL). The combined organic
extracts were washed with sat. NaCl (20 mL), then dried over anhy-
drous Na2SO4, filtered and evaporated under vacuum to give 14 (54
mg, 77%) as a white solid. The crude product was used in the next
step without further purification.
1H NMR (500 MHz, CDCl3): d = 8.72 (s, 1 H, NHCOCH2), 8.20 (d,
J = 9.2 Hz, 2 H, ArH, p-NA), 7.74 (d, J = 9.1 Hz, 2 H, ArH, p-NA),
7.36–7.20 (m, 5 H, ArH, PhH), 4.95 (d, J = 8.3 Hz, 1 H, NHCH),
4.27–4.18 (m, 1 H, NHCH), 2.97 (dd, J = 13.8, 7.3 Hz, 1 H, CHH-
Ph), 2.90 (dd, J = 13.8, 7.2 Hz, 1 H, CHHPh), 2.72 (dd, J = 15.7, 4.7
Hz, 1 H, CHHCO), 2.64 (dd, J = 15.8, 7.7 Hz, 1 H, CHHCO), 1.39
[s, 9 H, C(CH3)3].
13C NMR (151 MHz, CDCl3): d = 169.5, 156.4, 143.9, 143.5, 137.0,
129.2, 128.8, 127.0, 125.0, 119.1, 80.5, 49.3, 42.7, 41.1, 28.3.
HRMS (ESI): m/z [M + H]+ calcd for C21H26N3O5: 400.1872; found:
400.1863.
Mp 164–168 °C; [a]D21 +73.9 (c 1.23, CH3OH).
IR (KBr): 3448, 3424, 3348 sh, 3059, 2927, 2853, 1722, 1618,
1559, 1260, 1222, 1077, 766, 704 cm–1.
1H NMR (300 MHz, CD3OD): d = 7.42–7.26 (m, 5 H, ArH), 5.49
(dd, J = 28.2, 3.8 Hz, 1 H, NCH), 5.25 (dd, J = 49.3, 3.8 Hz, 1 H,
CHF), 2.00 (s, 3 H, CCH3).
13C NMR (75 MHz, CD3OD): d = 172.7, 170.6 (d, J = 24 Hz),
137.4, 129.6, 129.5, 129.3, 91.3 (d, J = 189 Hz), 55.9 (d, J = 19 Hz),
22.5.
19F NMR (282.4 MHz, CD3OD): d = –200.9 (dd, J = 49, 28 Hz).
tert-BuOSuc-(S)-b3-hPhe p-Nitrophenyl Ester (16a)
N-Boc-protected compound 15a (136 mg, 0.34 mmol) was dis-
solved in 10% TFA in CH2Cl2 (5 mL). The reaction mixture was
stirred at r.t. for 18 h, then concentrated in vacuo to give the free
amine as the TFA salt. The amine, succinic acid mono-tert-butyl es-
ter (71 mg, 0.41 mmol), EDCI (79 mg, 0.41 mmol), and HOAt (56
mg, 0.41 mmol) were dissolved in anhydrous CH2Cl2 (4 mL) under
N2. Et3N (0.19 mL, 1.3 mmol) was added and the reaction mixture
was stirred at r.t. for 18 h. The reaction mixture was partitioned be-
tween EtOAc (5 mL) and aq HCl (0.5 M, 5 mL). The organic phase
was separated and washed sequentially with aq HCl (0.5 M, 5 mL)
and sat. aq NaCl (5 mL). The organic phase was dried over MgSO4,
filtered and concentrated in vacuo. The crude product was purified
by radial chromatography (silica; 2 mm plate; EtOAc–CH2Cl2,
1:9→1:4) to give 16a (31 mg, 20%) as an off-white solid.
HRMS (ESI): m/z [M + H]+ calcd for C11H13FNO3: 226.0879;
found: 226.0873.
Boc-(S)-b3-hPhe p-Nitrophenyl Ester (15a)
Boc-(S)-b3-hPhe (252 mg, 0.90 mmol), p-nitroaniline (642 mg, 4.5
mmol), HATU (688 mg, 1.8 mmol) and HOAt (246 mg, 1.8 mmol)
were dissolved in anhydrous CH2Cl2 (2 mL) under N2. Et3N (0.51
mL, 3.6 mmol) was added and the reaction mixture was stirred at r.t.
for 18 h. The reaction mixture was partitioned between EtOAc (5
mL) and aq HCl (0.5 M, 5 mL). The organic phase was separated
and washed sequentially with aq HCl (0.5 M, 5 mL) and sat. aq
NaCl (5 mL). The organic phase was dried over MgSO4, filtered and
concentrated in vacuo. The crude product was recrystallized from
isopropanol, filtered and washed with ice-cold isopropanol until the
Mp 155 °C (dec.).
IR (KBr): 3312, 2979, 1653, 1557, 1511, 1342, 1152, 909, 733
cm–1.
1H NMR (500 MHz, CDCl3): d = 9.16 (br s, 1 H, NHAr), 8.17 (d,
J = 9.1 Hz, 2 H, ArH, p-NA), 7.79 (d, J = 9.1 Hz, 2 H, ArH, p-NA),
7.33–7.19 (m, 5 H, ArH, PhH), 6.37 (d, J = 8.1 Hz, 1 H, NHCH),
4.55–4.45 (m, 1 H, NHCH), 2.97 (d, J = 7.1 Hz, 2 H, CH2Ph), 2.74
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis and Inhibition of a-Chymotrypsin
1855
(dd, J = 15.1, 4.0 Hz, 1 H, CHCHHCO), 2.68–2.55 (m, 2 H,
CHCHHCO, CHHCH2), 2.50–2.42 (m, 1 H, CHHCH2), 2.40–2.33
(m, 2 H, CH2CH2), 1.40 [s, 9 H, C(CH3)3].
13C NMR (126 MHz, CDCl3): d = 172.7, 172.6, 169.7, 144.2, 143.3,
137.1, 129.1, 128.8, 127.0, 124.9, 119.3, 81.3, 48.3, 42.1, 40.0,
31.2, 30.6, 28.0.
3 H, PhCH2CH2, CHCHHPh), 3.10–2.94 (m, 2 H, PhCH2CH2), 2.75
(dd, J = 13.4, 9.5 Hz, 1 H, CHCHHPh).
13C NMR (75 MHz, CDCl3): d = 172.4, 153.4, 140.4, 135.2, 129.4,
128.9, 128.6, 128.5, 127.3, 126.3, 66.2, 55.1, 37.8, 37.1, 30.2.
HRMS (ESI): m/z [M + H]+ calcd for C19H20NO3: 310.1443; found:
310.1450.
HRMS (ESI): m/z [M + H]+ calcd for C24H30N3O6: 456.2135; found:
456.2122.
(R)-4-Benzyl-3-[(R)-2-fluoro-3-phenylpropanoyl]oxazolidin-2-
one (18)
tert-BuOSuc-(R)-b3-hPhe p-Nitrophenyl Ester (16b)
n-BuLi (21.0 mL, 34 mmol, 1.6 M in hexane) was added to a solu-
tion of freshly distilled i-Pr2NH (4.71 mL, 34 mmol) in anhydrous
THF (80 mL) at –78 °C under N2. The reaction was stirred at –78 °C
for 1 h, then a solution of 17 (10.0 g, 32 mmol) in anhydrous THF
(50 mL) under N2 was added. The mixture was stirred for 1 h at
–78 °C and then NFSI (10.6 g, 34 mmol) in anhydrous THF (30 mL)
under N2 was added dropwise. The resulting yellow mixture was
stirred for 2.5 h at –78 °C then for 2 h at 0 °C. Sat. aq NH4Cl (20
mL) was added to quench the reaction and the aqueous layer was ex-
tracted with CH2Cl2 (3 × 50 mL). The combined organic phases
were dried over MgSO4 and the solvent removed in vacuo to give
an orange oil. PE–EtOAc (4:1) was added to give a white precipitate
that was removed by filtration. The filtrate was concentrated in vac-
uo to give an orange oil that was purified by flash chromatography
on silica (PE–EtOAc, 4:1) to give 18 (7.98 g, 76%) as an off-white
solid with a diastereomeric ratio of 23:1 (determined by 19F NMR).
N-Boc-protected compound 15b (165 mg, 0.40 mmol) was dis-
solved in 10% TFA in CH2Cl2 (5 mL). The reaction mixture was
stirred at r.t. for 18 h, then concentrated in vacuo to give the free
amine as the TFA salt. The amine, succinic acid mono-tert-butyl es-
ter (84 mg, 0.48 mmol), EDCI (92 mg, 0.48 mmol) and HOAt (66
mg, 0.48 mmol) were dissolved in anhydrous CH2Cl2 (4 mL) under
N2. Et3N (0.22 mL, 1.6 mmol) was added and the reaction mixture
was stirred at r.t. for 18 h. The reaction mixture was partitioned be-
tween EtOAc (5 mL) and aq HCl (0.5 M, 5 mL). The organic phase
was separated and washed sequentially with aq HCl (0.5 M, 5 mL)
and sat. aq NaCl (5 mL). The organic phase was dried over MgSO4,
filtered and concentrated in vacuo. The crude product was purified
by radial chromatography (silica; 2 mm plate; EtOAc–CH2Cl2,
1:9→1:4) to give 16b (127 mg, 69%) as an off-white solid.
Mp 152 °C (dec.).
Mp 98–99 °C; [a]D22 –71.2 (c 0.85, CHCl3).
IR (KBr): 3030, 2924, 1783, 1718, 1392, 1213, 700 cm–1.
IR (KBr): 3088, 2979, 1653, 1558, 1512, 1337, 1152, 911, 732
cm–1.
1H NMR (500 MHz, CDCl3): d = 9.25 (br s, 1 H, NHAr), 8.14 (d,
J = 6.2 Hz, 2 H, ArH, p-NA), 7.78 (d, J = 6.2 Hz, 2 H, ArH, p-NA),
7.33–7.17 (m, 5 H, ArH, PhH), 6.48 (d, J = 6.7 Hz, 1 H, NHCH),
4.54–4.44 (m, 1 H, NHCH), 2.96 (d, J = 4.9 Hz, 2 H, CH2Ph), 2.76–
2.32 [m, 6 H, CHCH2CO, (CH2)2], 1.39 [s, 9 H, C(CH3)3)].
1H NMR (500 MHz, CDCl3): d = 7.36–7.19 (m, 10 H, ArH), 6.12
(ddd, J = 49.53, 8.61, 3.50 Hz, 1 H, CHF), 4.64–4.57 (m, 1 H,
NCH), 4.26–2.17 (m, 2 H, CHCH2O), 3.35 (dd, J = 13.5, 3.2 Hz,
1 H, CHCHHPh), 3.29–3.06 (m, 2 H, PhCH2CH2), 2.83 (dd,
J = 13.5, 9.6 Hz, 1 H, CHCHHPh).
13C NMR (126 MHz, CDCl3): d = 172.6, 172.5, 169.8, 144.2, 143.2,
137.1, 129.1, 128.7, 126.9, 124.9, 119.2, 81.2, 48.3, 41.9, 39.9,
31.1, 30.6, 28.0.
13C NMR (75 MHz, CDCl3): d = 168.9 (d, J = 22.9 Hz), 152.8,
135.1, 134.6, 129.3, 129.2, 128.8, 128.3, 127.3, 127.0, 89.3 (d,
J = 182.0 Hz), 66.9, 55.0, 38.5 (d, J = 22.2 Hz), 37.1.
HRMS (ESI): m/z [M + H]+ calcd for C24H30N3O6: 456.2135; found:
456.2115.
19F NMR (282 MHz, CDCl3): d = –190.76 (ddd, J = 49.7, 34.1, 19.7
Hz), –191.40 (ddd, J = 49.7, 35.3, 20.8 Hz).
HRMS (ESI): m/z [M + H]+ calcd for C19H19NO3F: 328.1349;
found: 328.1351.
(R)-4-Benzyl-3-(3-phenylpropanoyl)oxazolidin-2-one (17)
Oxalyl chloride (6.28 mL, 73 mmol) was added dropwise to a solu-
tion of 3-phenylpropanoic acid (10.0 g, 67 mmol) in anhydrous
CH2Cl2 (100 mL) under N2 at 0 °C. A catalytic amount of DMF was
added and the mixture was warmed to r.t. and stirred for 18 h. The
solvent was removed in vacuo to give crude 3-phenylpropionyl
chloride (11.30 g, quantitative yield) as a light-yellow oil.
(R)-4-Benzyl-3-[(R)-2-benzyl-3-(benzyloxy)-2-fluoro-
propanoyl]oxazolidin-2-one (19)
TiCl4 (2.25 mL, 21 mmol) was added to a solution of 18 (5.63 g, 17
mmol) in anhydrous CH2Cl2 (75 mL) at r.t. under N2. The mixture
was stirred for 5 min, cooled to 0 °C and stirring was continued for
10 min at 0 °C. DIPEA (3.30 mL, 19 mmol) was added and the mix-
ture was stirred for 1 h at 0 °C. Benzyl chloromethyl ether (8.55
mL, 61 mmol) was added and stirring was continued for 1 h at 0 °C,
followed by 3 h at r.t. Sat. aq NH4Cl (20 mL) was added to quench
the reaction and the aqueous layer was extracted with CH2Cl2
(2 × 50 mL) and Et2O (1 × 50 mL). The combined organic phases
were dried over MgSO4 and then concentrated in vacuo to give a
dark-red oil that was purified by flash chromatography on silica
(PE–EtOAc, 9:1→7:3). The pure product 19 was isolated as a white
solid (4.00 g, 52%), >95% de (by 19F and 1H NMR).
n-BuLi (40 mL, 25 mmol, 1.6 M in hexane) was added dropwise to
a solution of (4R)-4-benzyl-2-oxazolidinone (4.25 g, 24 mmol) in
anhydrous THF (70 mL) at –78 °C under N2. After 1 h, a solution of
3-phenylpropionyl chloride (4.49 g, 27 mmol) in anhydrous THF
(25 mL) under N2 was added dropwise at –78 °C. The reaction mix-
ture was then allowed to warm to r.t. and stirred for 18 h. The reac-
tion mixture was quenched by the addition of sat. aq NaCl (20 mL)
and the resulting mixture was concentrated in vacuo. The residue
was partitioned between CH2Cl2 (50 mL) and H2O (50 mL) and the
aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The com-
bined organic layers were dried over MgSO4, filtered and concen-
trated in vacuo. Recrystallization from hot PE–EtOAc (4:1) gave 17
(5.35 g, 96%) as white needles.
Mp 67–69 °C; [a]D22 –62 (c 1.13, CHCl3).
IR (KBr): 3426, 3030, 2922, 1782, 1709, 1352, 1210, 1117, 736,
700 cm–1.
Mp 100–101 °C; [a]D22 –51.2 (c 0.95, CHCl3).
IR (KBr): 3032, 1780, 1697, 1389, 1211, 702 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.40–7.10 (m, 10 H, ArH), 4.70–
463 (m, 1 H, NCH), 4.25–4.06 (m, 2 H, CHCH2O), 3.39–3.18 (m,
1H NMR (500 MHz, CDCl3): d = 7.34–7.06 (m, 15 H, ArH), 4.62–
4.57 (m, 1 H, NCH), 4.57 (dd, J = 11.8 Hz, 1 H, OCHHPh), 4.44
(dd, J = 11.8 Hz, 1 H, OCHHPh), 4.37 (dd, J = 33.4, 11.2 Hz, 1 H,
CFCHHO), 3.99 (dd, J = 9.0, 2.6 Hz, 1 H, CHCHHO), 3.92 (dd,
J = 8.2, 8.2 Hz, 1 H, CHCHHO), 3.75 (dd, J = 17.4, 11.2 Hz, 1 H,
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

1856
V. Peddie et al.
SPECIAL TOPIC
19F NMR (282 MHz, CDCl3): d = –167.7, –168.2 (m).
HRMS (ESI): m/z [M + H]+ calcd for C17H17O3F: 311.1059; found:
311.1073.
CF-CHHO), 3.62 (dd, J = 30.2, 14.6 Hz, 1 H, PhCHHCF), 3.36 (dd,
J = 24.8, 14.6 Hz, 1 H, PhCHHCF), 2.84 (dd, J = 13.4, 2.9 Hz, 1 H,
CHCHHPh), 2.36 (dd, J = 13.3, 10.3 Hz, 1 H, CHCHHPh).
13C NMR (75 MHz, CDCl3): d = 168.4 (d, J = 27.0 Hz), 152.2,
137.5, 135.3, 134.0, 130.3, 129.3, 128.9, 128.4, 128.4, 127.8, 127.8,
127.4, 127.3, 100.9 (d, J = 190.5 Hz), 73.8, 72.4 (d, J = 22.9 Hz),
66.6, 56.9, 39.0 (d, J = 21.5 Hz), 37.6.
(S)-2-Benzyl-3-(benzyloxy)propanoic Acid (22)
A 50% aqueous solution of H2O2 (5.30 mL, 92 mmol) was added
dropwise to a solution of 20 (3.94 g, 9.2 mmol) in THF–H2O (4:1,
150 mL) at 0 °C. LiOH·nH2O (769 mg, 18 mmol) was added por-
tionwise. The mixture was warmed to r.t., stirred for 3 h and then re-
cooled to 0 °C. Aqueous Na2SO3 (1.0 M, 20 mL) was added and the
mixture was partitioned between CH2Cl2 (100 mL) and H2O. The
aqueous phase was acidified to pH 2 with aq HCl (1 M) and extract-
ed with EtOAc (3 × 50 mL). The combined EtOAc layers were
dried over Na2SO4 and concentrated in vacuo to give 22 (2.16 g,
87%) as a colorless oil, which was used without further purification.
[a]D22 –9.0 (c 1.03, CHCl3).
IR (KBr): 3030, 2928, 1713, 1455, 1273, 1116, 740, 699 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.41–7.09 (m, 10 H, ArH), 4.53
(d, J = 12.1 Hz, 1 H, OCHHPh), 4.49 (d, J = 12.1 Hz, 1 H, OCHH-
Ph), 3.64–3.57 (m, 2 H, CHCH2O), 3.06–2.95 (m, 2 H, PhCHHCH,
PhCH2CH), 2.88 (dd, J = 12.8, 6.7 Hz, 1 H, PhCHHCH).
19F NMR (282 MHz, CDCl3): d = –150.7, –152.4 (m).
HRMS (ESI): m/z [M + H]+ calcd for C27H27NO4F: 448.1924;
found: 448.1927.
(R)-4-Benzyl-3-[(S)-2-benzyl-3-(benzyloxy)propanoyl]oxazoli-
din-2-one (20)
TiCl4 (1.24 mL, 11 mmol) was added to a solution of 17 (2.92 g, 9.4
mmol) in anhydrous CH2Cl2 (40 mL) at r.t. under N2. The mixture
was stirred for 5 min, cooled to 0 °C, and stirring was continued for
10 min at 0 °C. DIPEA (1.45 mL, 10 mmol) was added and the mix-
ture was stirred for 1 h at 0 °C. Benzyl chloromethyl ether (4.70
mL, 38 mmol) was added and stirring was continued for 1 h at 0 °C,
followed by 3 h at r.t. Sat. aq NH4Cl (10 mL) was added to quench
the reaction and the aqueous layer was extracted with CH2Cl2
(3 × 25 mL) and Et2O (25 mL). The combined organic phases were
dried over MgSO4 and concentrated in vacuo to give an orange oil
that was purified by flash chromatography (silica; PE–EtOAc, 7:3).
The pure product 20 was isolated as a colorless oil (3.96 g, 98%),
>95% de (determined by 1H NMR).
13C NMR (126 MHz, CDCl3): d = 179.0, 138.4, 137.7, 128.9, 128.5,
128.4, 127.7, 127.7, 126.5, 73.2, 69.3, 47.4, 34.2.
HRMS (ESI): m/z [M + H]+ calcd for C17H19O3: 271.1334; found:
271.1328.
[a]D –73.0 (c 1.19, CHCl3).
(R)-Methyl 2-Benzyl-3-benzyloxy-2-fluoropropanoate (23)
The acid 21 (912 mg, 3.1 mmol) was esterified using General Pro-
cedure B. The crude product was purified by flash chromatography
on silica (EtOAc–PE, 3:7) to give 23 (929 mg, 97%) as a colorless
oil.
IR (KBr): 3379, 3032, 2923, 1782, 1697, 1389, 1211, 1103, 1049,
741, 702 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.45–7.07 (m, 15 H, ArH), 4.60–
4.50 (m, 4 H, NCH, CHCO, OCH2Ph), 4.02 (ddd, J = 8.9, 2.7, 1.3
Hz, 1 H, NCHCHHO), 3.94–3.81 (m, 1 H, NCHCHHO,
CHHOCH2Ph), 3.66 (ddd, J = 9.3, 4.8, 1.2 Hz, 1 H, CHHOCH2Ph),
3.18 (dd, J = 13.6, 3.2 Hz, 1 H, NCHCHHPh), 2.97 (dd, J = 13.0,
8.5 Hz, 1 H, PhCHHCHCO), 2.88 (dd, J = 13.3, 7.2 Hz, 1 H, PhCH-
HCHCO), 2.68 (dd, J = 13.4, 9.5 Hz, 1 H, NCHCHHPh).
13C NMR (75 MHz, CDCl3): d = 174.1, 153.0, 138.4, 138.0, 135.2,
129.4, 129.0, 128.8, 128.3, 128.3, 127.6, 127.5, 127.1, 126.4, 73.1,
70.5, 65.8, 55.2, 45.1, 37.7, 35.1.
[a]D22 +2.0 (c 1.0, CHCl3).
IR (KBr): 3407, 3032, 2954, 1767, 1740, 1455, 1204, 1097, 745,
700 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.37–7.15 (m, 10 H, ArH), 4.64
(d, J = 12.2 Hz, 1 H, OCHHPh), 4.55 (d, J = 12.2 Hz, 1 H, OCHH-
Ph), 3.86 (dd, J = 27.0, 11.0 Hz, 1 H, CFCHHO), 3.74–3.64 (m,
1 H, CFCHHO), 3.70 (d, J = 1.7 Hz, 3 H, OCH3), 3.15 (d, J = 22.9
Hz, 2 H, PhCH2CF).
13C NMR (75 MHz, CDCl3): d = 169.9 (d, J = 24.7 Hz), 137.5,
133.7, 130.0, 128.4, 128.6, 127.8, 127.7, 127.3, 97.6 (d, J = 194.2
Hz), 73.7, 72.4 (d, J = 21.6 Hz), 52.5, 39.9 (d, J = 21.3 Hz).
HRMS (ES): m/z [M + H]+ calcd for C27H28NO4: 430.2018; found:
430.2022.
(R)-2-Benzyl-3-benzyloxy-2-fluoropropanoic Acid (21)
A 50% aqueous solution of H2O2 (6.47 mL, 112 mmol) was added
dropwise to a solution of 19 (5.0 g, 11 mmol) in THF–H2O (4:1, 150
mL) at 0 °C. LiOH·H2O (940 mg, 22 mmol) was added portion-
wise. The mixture was warmed to r.t., stirred for 3 h, and then re-
cooled to 0 °C. Aqueous Na2SO3 (1.0 M, 20 mL) was added and the
mixture was partitioned between CH2Cl2 (100 mL) and H2O. The
aqueous phase was acidified to pH 2 with aq HCl (1 M) and extract-
ed with EtOAc (3 × 50 mL). The combined EtOAc layers were
dried over Na2SO4, and then concentrated in vacuo to give 21 (2.86
g, 87%) as a white solid.
19F NMR (282 MHz, CDCl3): d = –168.5, –168.8 (m).
HRMS (ESI): m/z [M + H]+ calcd for C18H20O3F: 303.1396; found:
303.1385.
(S)-Methyl 2-Benzyl-3-(benzyloxy)propanoate (24)
The acid 22 (1.82 g, 6.4 mmol) was esterified using General Proce-
dure B. The crude product was purified by flash chromatography
(silica; EtOAc–PE, 3:7) to give 24 (1.53 g, 84%) as a colorless oil.
[a]D22 +2.2 (c 1.18, CHCl3).
Mp 59–60 °C; [a]D22 +2.2 (c 0.91, CHCl3).
IR (KBr): 3031, 2928, 2584, 1737, 1454, 1099, 739, 700 cm–1.
IR (KBr): 3375, 3029, 2951, 1736, 1454, 1205, 1168, 1092, 746,
699 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.36–7.12 (m, 10 H, ArH), 4.51
(d, J = 12.5 Hz, 1 H, OCHHPh), 4.48 (d, J = 12.4 Hz, 1 H, OCHH-
Ph), 3.66–3.62 (m, 1 H, CHCHHO), 3.63 (s, 3 H, OCH3), 3.56 (dd,
J = 9.2, 5.0 Hz, 1 H, CHCHHO), 3.01–2.94 (m, 2 H, PhCHHCH,
PhCH2CH), 2.90–2.83 (m, 1 H, PhCHHCH).
13C NMR (126 MHz, CDCl3): d = 174.1, 138.7, 138.0, 128.9, 128.4,
128.3, 127.6, 127.6, 126.4, 73.1, 69.9, 51.7, 47.7, 34.6.
1H NMR (500 MHz, CDCl3): d = 8.00 (br s, 1 H, OH), 7.36–7.18
(m, 10 H, ArH), 4.63 (d, J = 12.2 Hz, 1 H, OCHHPh), 4.55 (d,
J = 12.2 Hz, 1 H, OCHHPh), 3.88 (dd, J = 28.1, 11.0 Hz, 1 H,
CFCHHO), 3.68 (dd, J = 15.1, 11.1 Hz, 1 H, CFCHHO), 3.21–3.12
(m, 2 H, PhCH2CF).
13C NMR (75 MHz, CDCl3): d = 173.7 (d, J = 27.2 Hz), 137.1,
133.2, 130.1, 130.0, 128.4, 127.9, 127.7, 127.4, 97.2 (d, J = 192.9
Hz), 73.8, 72.2 (d, J = 21.5 Hz), 39.5 (d, J = 21.0 Hz).
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis and Inhibition of a-Chymotrypsin
1857
HRMS (ESI): m/z [M + H]+ calcd for C18H2O3: 285.1491; found:
285.1494.
13C NMR (126 MHz, CDCl3): d = 168.1 (d, J = 25.1 Hz), 145.2,
132.5, 132.3, 129.9, 129.9, 128.6, 128.0, 127.7, 94.9 (d, J = 197.2
Hz), 70.3 (d, J = 23.3 Hz), 52.8, 39.6 (d, J = 21.4 Hz), 21.7.
(R)-Methyl 2-Benzyl-2-fluoro-3-hydroxypropanoate (25)
A catalytic amount of Pd/C was added to a solution of 23 (1.49 g,
4.9 mmol) in MeOH (20 mL) at r.t. The apparatus was evacuated
and flushed three times with H2 and stirred at r.t. for 8 h under an H2
atmosphere. The Pd/C catalyst was removed by filtration through
Celite and the filtrate was concentrated in vacuo to give 25 as a col-
orless oil (1.00 g, 95%).
19F NMR (282 MHz, CDCl3): d = –168.4, –168.7 (m).
HRMS (ESI): m/z [M + H]+ calcd for C18H20O5FS: 367.1015; found:
367.1015.
(S)-Methyl 2-Benzyl-3-tosyloxypropanoate (28)
Et3N (1.29 mL, 9.3 mmol), 4-(dimethylamino)pyridine (cat.) and
tosyl chloride (973 mg, 5.1 mmol), were dissolved in a minimum
amount of anhydrous CH2Cl2 under N2 and added dropwise to a so-
lution of 26 (901 mg, 4.6 mmol) in anhydrous CH2Cl2 (60 mL), at
0 °C, under N2. The mixture was warmed to r.t., stirred for 10 h, and
then re-cooled to 0 °C. H2O (60 mL) was added to quench the reac-
tion and the aqueous layer was extracted with CH2Cl2 (3 × 60 mL).
The combined organic phases were washed with aq HCl (5%, 150
mL) and H2O (100 mL), dried over Na2SO4 and concentrated in vac-
uo to give the crude product as a pale-yellow solid. The crude prod-
uct was purified by flash chromatography (silica; EtOAc–PE, 1:4)
to give 28 (1.60 g, 99%) as a white solid.
Mp 33–35 °C; [a]D22 +4.9 (c 1.12, CHCl3).
IR (KBr): 2955, 1744, 1366, 1180, 980, 818, 556 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.75 (d, J = 8.3 Hz, 2 H, ArH,
OTs), 7.34 (d, J = 8.5 Hz, 2 H, ArH, OTs), 7.27–7.18 (m, 3 H, ArH,
PhH), 7.05 (d, J = 6.8 Hz, 2 H, ArH, PhH), 4.16–4.08 (m, 2 H,
CH2OTs), 3.59 (s, 3 H, OCH3), 3.02–2.93 (m, 2 H, PhCHHCH,
PhCH2CH), 2.85–2.78 (m, 1 H, PhCHHCH), 2.45 (s, 3 H, CCH3).
[a]D22 –11.5 (c 1.17, CHCl3).
IR (KBr): 3425, 3032, 2932, 1747, 1636, 1497, 1439, 1205, 1094,
1042, 745, 702 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.39–7.21 (m, 5 H, ArH), 4.09–
3.82 (m, 2 H, CH2OH), 3.76 (d, J = 2.5 Hz, 3 H, OCH3), 3.27–3.15
(m, 2 H, PhCH2), 2.46 (br s, 1 H, OH).
13C NMR (75 MHz, CDCl3): d = 170.1 (d, J = 25.7 Hz), 133.5,
130.0, 128.4, 127.3, 97.9 (d, J = 190.6 Hz), 65.8 (d, J = 23.5 Hz),
52.5, 39.5 (d, J = 21.2 Hz).
19F NMR (282 MHz, CDCl3): d = –171.8, –172.1 (m).
HRMS (ESI): m/z [M + H]+ calcd for C11H14O3F: 213.0927; found:
213.0919.
(S)-Methyl 2-Benzyl-3-hydroxypropanoate (26)
A catalytic amount of Pd/C was added to a solution of 24 (1.40 g,
4.9 mmol) in MeOH (20 mL) at r.t. The apparatus was evacuated
and flushed three times with H2 and the reaction mixture was stirred
at r.t. for 8 h under an H2 atmosphere. The Pd/C catalyst was re-
moved by filtration through Celite and the filtrate was concentrated
in vacuo to give 26 as a colorless oil (943 mg, 98%).
13C NMR (126 MHz, CDCl3): d = 172.0, 144.9, 137.1, 132.6, 129.8,
128.8, 128.6, 127.8, 126.8, 68.6, 52.0, 46.4, 34.0, 21.6.
HRMS (ESI): m/z [M + H]+ calcd for C18H21O5S: 349.1110; found:
349.1116.
[a]D22 –28.4 (c 1.18, CHCl3) {Lit.11 [a]D –25.1 (c 0.56, CHCl3)}.
IR (KBr): 3460, 3054, 1734, 1265, 738, 704 cm–1.
(R)-Methyl 3-Azido-2-benzyl-2-fluoropropanoate (29)
NaN3 (1.58 g, 24 mmol) was added to a solution of 27 (1.78 g, 4.9
mmol) in anhydrous DMF (40 mL) at r.t. The mixture was heated to
80 °C and stirred for 36 h under N2 and then cooled to r.t. EtOAc
(50 mL) and H2O (25 mL) were added, the organic layer was sepa-
rated and extracted with H2O (2 × 50 mL) and sat. aq NaCl (50 mL),
and dried over MgSO4. The organic layer was concentrated in vacuo
and the crude product was purified by radial chromatography (sili-
ca; 5 mm plate; EtOAc–PE, 3:17) to give 29 (511 mg, 45%) as a col-
orless oil.
1H NMR (500 MHz, CDCl3): d = 7.31–7.16 (m, 5 H, ArH), 3.77–
3.67 (m, 2 H, CH2OH), 3.69 (s, 3 H, OCH3), 3.02 (dd, J = 16.8, 9.9
Hz, 1 H, PhCHHCH), 2.89–2.83 (m, 2 H, PhCHHCH, PhCH2CH),
2.17 (br s, 1 H, OH).
13C NMR (126 MHz, CDCl3): d = 175.1, 138.5, 128.9, 128.5, 126.5,
62.2, 51.9, 49.0, 34.4.
HRMS (ESI): m/z [M + H]+ calcd for C11H15O3: 195.1021; found:
195.1020.
[a]D22 +50.9 (c 1.18, CHCl3).
(R)-Methyl 2-Benzyl-2-fluoro-3-tosyloxypropanoate (27)
Et3N (0.95 mL, 6.8 mmol), 4-(dimethylamino)pyridine (cat.) and
tosyl chloride (652 mg, 3.4 mmol), were dissolved in a minimum
amount of anhydrous CH2Cl2 under N2 and added dropwise to a so-
lution of 25 (660 mg, 3.1 mmol) in anhydrous CH2Cl2 (50 mL), at
0 °C, under N2. The mixture was warmed to r.t., stirred for 10 h, and
then re-cooled to 0 °C. H2O (50 mL) was added to quench the reac-
tion and the aqueous layer was extracted with CH2Cl2 (3 × 50 mL).
The combined organic phases were washed with aq HCl (5%, 150
mL) and H2O (100 mL), dried over Na2SO4 and concentrated in vac-
uo to give the crude product as a pale-yellow solid. Recrystalliza-
tion from a minimal amount of PE–EtOAc (4:1), gave 27 (700 mg,
79%) as a white solid.
IR (KBr): 3033, 2955, 2108, 1767, 1742, 1441, 1277, 1223, 1096,
702 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.35–7.15 (m, 5 H, ArH), 3.73 (d,
J = 2.4 Hz, 3 H, OCH3), 3.71–3.49 (m, 2 H, CH2N3), 3.23–3.13 (m,
2 H, PhCH2).
13C NMR (75 MHz, CDCl3): d = 169.3 (d, J = 25.4 Hz), 133.0,
130.0, 128.5, 127.6, 97.0 (d, J = 195.6 Hz), 55.3 (d, J = 22.1 Hz),
52.8, 40.7 (d, J = 21.0 Hz).
19F NMR (282 MHz, CDCl3): d = –166.8, –167.1 (m).
HRMS (ESI): m/z [M + H]+ calcd for C11H13N3O2F: 238.0992;
found: 238.0982.
Mp 94–96 °C; [a]D22 –7.9 (c 1.11, CHCl3).
IR (KBr): 1771, 1744, 1367, 1192, 1178, 995, 553 cm–1.
(S)-Methyl 3-Azido-2-benzylpropanoate (30)
NaN3 (1.06 mg, 17 mmol) was added to a solution of 28 (1.14 g, 3.3
mmol) in anhydrous DMSO (25 mL) at r.t. The mixture was stirred
for 6 h under N2 at r.t. EtOAc (30 mL) and H2O (15 mL) were added
and the organic layer was separated and extracted with H2O (3 × 30
mL), with sat. aq NaCl (30 mL) and dried over NaSO4. The organic
layer was concentrated in vacuo and the crude product was purified
1H NMR (500 MHz, CDCl3): d = 7.77 (d, J = 8.3 Hz, 2 H, ArH,
OTs), 7.35 (d, J = 8.5 Hz, 2 H, ArH, OTs), 7.29–7.25 (m, 4 H, ArH,
PhH), 7.14–7.10 (m, 1 H, ArH, PhH), 4.37 (dd, J = 23.4, 11.0 Hz,
1 H, CHHOTs), 4.23 (dd, J = 16.3, 11.0 Hz, 1 H, CHHOTs), 3.65
(s, 3 H, OCH3), 3.14 (d, J = 22.1 Hz, 1 H, PhCH2), 2.46 (s, 3 H,
CCH3).
Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

1858
V. Peddie et al.
SPECIAL TOPIC
by radial chromatography (silica; 5 mm plate; EtOAc–PE, 1:9) to
give 30 (454 mg, 64%) as a colorless oil.
substrate concentrations with 0.011 mg/mL and 43.7 m/mL bCT, re-
spectively.
[a]D22 +11.2 (c 1.05, CHCl3).
IR (KBr): 2955, 2106, 1736, 1450, 1196, 748, 702 cm–1.
Assay of Human a-Chymotrypsin Activity
Activity of hCT was analyzed on a THERMOmax microplate read-
er recording the absorbance at 405 nm at 25 0.1 °C in bovine se-
rum albumin-coated 200 mL well plates. Assay buffer was:
phosphate (100 mM), NaCl (500 mM), pH 7.8. Stock solutions of
the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA (20 mM) and
the inhibitors 1a and 5 (440 mM) were prepared in DMSO and
stored at –20 °C; hCT was dissolved in assay buffer (500 mg/mL)
and stored at –80 °C. Determination of the Michaelis constant for
the substrate Suc-Ala-Ala-Pro-Phe-pNA at seven concentrations
(15–240 mM) was undertaken with 0.125 mg/mL hCT and 5% v/v
DMSO. Inhibition of hCT was determined at two substrate concen-
trations 7.2 mM (0.2 × Km) and 14.4 mM (0.4 × Km) Suc-Ala-Ala-
Pro-Phe-pNA, in 5% DMSO with 0.050 mg/mL hCT, and seven dif-
ferent inhibitor concentrations (0.5–8.0 mM) when soluble. Control
experiments were performed in the absence of inhibitor and without
both inhibitor and enzyme to determine uninhibited enzyme activity
and non-enzymatic hydrolysis of substrate, respectively. Progress
curves over 10 min were characterized by a linear steady-state turn-
over of the substrate. Ki values and modes of inhibition were ob-
tained according to Dixon40 from duplicate experiments.
1H NMR (500 MHz, CDCl3): d = 7.33–7.13 (m, 5 H, ArH), 3.70 (s,
3 H, OCH3), 3.50 (dd, J = 12.2, 7.3 Hz, 1 H, CHHN3), 3.44 (dd,
J = 12.3, 4.8 Hz, 1 H, CHHN3), 3.03 (dd, J = 12.7, 5.9 Hz, 1 H, Ph-
CHHCH), 2.91–2.81 (m, 2 H, PhCHHCH, PhCH2CH).
13C NMR (126 MHz, CDCl3): d = 173.3, 137.7, 128.9, 128.6, 126.8,
52.0, 51.4, 46.9, 35.3.
HRMS (ESI): m/z [M + Na]+ calcd for C11H13N3O2Na: 242.0905;
found: 242.0908.
Assay of Bovine a-Chymotrypsin Activity
The activity of bCT was assayed spectrophotometrically with a
Varian Cary 5000 UV-VIS-NIR spectrophotometer equipped with
a thermostated multicell holder at 25.0 0.1 °C. Assay buffer was:
Tris-HCl (77 mM), CaCl2 (20 mM), pH 7.8 (pH optimum of a-
chymotrypsin68). A solution of bCT (21.9 mg/mL) in aq HCl (1 mM)
was prepared daily by a 1:40 dilution of a stock solution (874 mg/
mL) in aq HCl (1 mM) and kept at 0 °C. A 1:100 dilution in ice-cold
aq HCl (1 mM) was prepared immediately before starting each mea-
surement. Stock solutions of the substrate Suc-Ala-Ala-Pro-Phe-
pNA (10 mM) and all inhibitors (10–50 mM) were freshly prepared
in DMSO and stored at r.t. Compound 10 was analyzed at a single
concentration (200 mM); for all other compounds five different in-
hibitor concentrations, [I], were used. Progress curves were moni-
tored at 405 nm over 6 min and characterized by a linear steady-
state turnover of the substrate.
Acknowledgment
V.P. thanks the Tertiary Education Commission of New Zealand for
funding. M.P. and A.D.A. gratefully acknowledge financial support
from ARC grant DP0771901. This work was supported by a fel-
lowship within the ‘Postdoc-Program’ of the German Academic
Exchange Service (M.P.). The authors wish to thank Prof. Dr. Ray-
mond J. Butcher from Howard University, Washington, D.C., USA
for solving the crystal structure of 4b.
Inhibition studies were performed in the presence of 6% v/v DMSO
in a volume of 1 mL containing 0.011 mg/mL bCT, different con-
centrations of Suc-Ala-Ala-Pro-Phe-pNA (10–100 mM) and inhibi-
tor. Into a cuvette containing 890 mL assay buffer, 10 mL of substrate
solution (1–10 mM), inhibitor stock, and DMSO were added to give
a total volume of 950 mL. After thoroughly mixing the contents of
the cuvette, the enzymatic reaction was initiated by adding 50 mL of
bCT solution. Uninhibited enzyme activity was determined by add-
ing DMSO instead of inhibitor solution. Non-enzymatic hydrolysis
of Suc-Ala-Ala-Pro-Phe-pNA was analyzed by adding DMSO and
1 mM aqueous HCl instead of inhibitor and enzyme solution, re-
spectively, and found to be negligible. The rate of enzyme-cata-
lyzed hydrolysis of 100 mM substrate was determined without in-
hibitor in each experiment and was set to 100%. Ki values of all
inhibitors (except of 3a) were determined graphically according to
Dixon40 using mean values of percentage rates obtained in three or
four separate experiments at two different substrate concentrations,
[S]. The mode of inhibition was concluded from Dixon plots40 and
plots [S]/v versus [I] according to Cornish-Bowden,41 where v is the
enzyme activity. For compound 3a, an IC50 value was obtained from
triplicate experiments at a single substrate concentration (50 mM)
using the equation v = v0/(1 + [I]/IC50), were v0 and v are the rates in
the absence and presence of inhibitor, respectively. The Ki value of
3a was calculated according to Cheng and Prusoff,42 and Chou43 us-
ing the equation Ki = IC50/(1 + [S]/Km), where [S] and Km are the
substrate concentration and the Michaelis constant, respectively.
For this purpose, the parameter Km of Suc-Ala-Ala-Pro-Phe-pNA
was determined in the absence of inhibitor. The obtained value of
67 4 mM is in good agreement with data reported in literature.69–71
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Synthesis 2010, No. 11, 1845–1859 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis and Inhibition of a-Chymotrypsin
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