Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 200
8
Supporting Information for
Biosynthesis and stability of coiled-coil peptides
containing
(2
S
,4
R
)-5,5,5-trifluoroleucine and (2
S
,4
S
)-5,5,5-trifluoroleucine
Jin Kim Montclare,
[b]+
Soojin Son,
[a]+
Ginevra A. Clark,
[c]
Krishna Kumar,
[c, d]
and David
A. Tirrell
[a]
*
[a] Dr. S. Son, Prof. D. A. Tirrell
Division of Chemistry and Chemical Engineering
California Institute of Technology
Pasadena, CA 91125, USA
Fax: (+1) 626-793-8472
E-mail:
tirrell@caltech.edu
[b] Prof. J. K. Montclare
Department of Chemical and Biological Sciences
Polytechnic University
Brooklyn, NY 11201, USA
Department of Biochemistry
SUNY Downstate Medical Center
Brooklyn, NY 11203
[c] G. A. Clark, Prof. K. Kumar
Department of Chemistry
Tufts University
Medford, MA 02155, USA
[d] Prof. K. Kumar
Cancer Center
Tufts-New England Medical Center
Boston, MA 02110, USA
+ These authors contributed equally.
Keywords:
Non-canonical amino acids • protein engineering • t
hermostability •
biosynthesis • stereochemical control
General Procedures
Flash column chromatography was performed on Kieseg
el 60 silica gel (230-240 mesh,
EM Science). Analytical thin-layer chromatography
was performed using E. Merck
silica gel Kiesegel 60 F
254
(0.24 mm) plates. Compounds were made visible by
staining
with a ninhydrin solution followed by heating. Mas
s spectra were obtained on a Thermo
Finnigan LTQ ESI-MS. Solution of compounds in MeOH
:H
2
O:AcOH (9:0.9:0.1) were
introduced into the instrument by direct infusion.
Nuclear magnetic resonance spectra
were recorded on a Bruker DPX-300 instrument using
standard deuterated solvents.
Stereochemical Resolution of 3 and 4 from 2
The racemic mixture of
N
-Boc
t
-butyl esters of trifluoroleucine (TFL) could be ea
sily
separated into the two enantiomeric pairs
5a
and
5b
by flash column chromatography. In
addition, racemization at C2 allowed for recycling
of the undesired 2
R
diastereomers
(Scheme S1). Assignment of absolute stereochemistr
y and corresponding
1
H NMR
spectra have been previously reported for compounds
3
and
4
.
1
Analytical data for new
compounds are reported below. Procedures for all ot
her compounds were adapted from
literature.
1
N
-Boc-5,5,5-TFL
t
-butyl esters
(
5a
and
5b
)
TFL (
2)
was converted to the
N
-Boc derivative
using literature procedures.
2
N
-Boc TFL
(2.05 g, 7.03 mmol) was added to anhydrous
t
-BuOH (100 mL) maintained at 32 °C. To
this solution, (Boc)
2
O (1.73 g, 7.93 mmol) and DMAP (88 mg, 0.72 mmol) w
ere added in
that order. The reaction was stirred under argon f
or 1 h. The product was purified by
flash chromatography using
n
-pentane/ methyl
t
-butyl ether (MTBE) (20:1) as the eluent
to give 560 mg of
5a
as a white solid and 561 mg of
5b
as a pale yellow oil. A mixture
of diastereomers (162 mg) was also recovered, resul
ting in an overall yield of 52% from
2
.
(2
S
,4
S
)-,(2
R
,4
R
)-
N
-Boc-5,5,5-trifluoroleucine
t
-butyl ester (5b)
R
f
: pentane/MTBE (5:1) 0.43;
1
H NMR (300 MHz, CDCl
3
)
δ
1.20-1.22 (d, 3H,
J
= 6.9
Hz), 1.45 (s, 9H), 1.47 (s, 9H), 1.76-1.84 (dt, 2H,
J
= 3.9, 9.9 Hz), 2.2-2.4 (m, 1H), 4.24-
4.26 (m, 1H), 4.94-4.97 (d, 1H,
J
= 8.1 Hz);
13
C NMR (75.5 MHz, CDCl
3
)
δ
171.9,
156.1, 130.4, 126.7, 82.89, 80.51, 51.72, 35.31 (q,
2
J
CF
= 26.9 Hz), 33.42, 30.13, 28.68,
28.36, 12.70; ESI-MS
m
/
z
364.20 (100, [M+23]
+
), calculated for C
15
H
26
NO
4
F
3
341.37.
(2
S
,4
R
)-,(2
R
,4
S
)-
N
-Boc-5,5,5-trifluoroleucine t-butyl ester (5a)
R
f
: pentane/MTBE (5:1) 0.38;
1
H NMR (300 MHz, CDCl
3
)
δ
1.18-1.20 (d, 3H,
J
= 6.9
Hz), 1.45 (s, 9H), 1.48 (s, 9H), 1.57-1.67 (m, 2H),
2.07-2.16 (m, 1H), 2.37-2.39 (m, 1H)
4.20-4.22 (m, 1H), 5.10-5.14 (d, 1H,
J
= 6.0 Hz);
13
C NMR (75.5 MHz, CDCl
3
)
δ
171.5,
155.5, 130.3, 126.6, 83.08, 80.43, 52.43, 35.45 (q,
2
J
CF
= 26.2 Hz), 33.64, 30.12, 28.70,
28.37, 13.36; ESI-MS:
m
/
z
364.17 (100, [M+23]
+
), calcd for C
15
H
26
NO
4
F
3
341.37.
(2
S
,4
R
)-5,5,5-trifluoroleucine (4)
To a solution of
5a
(622 mg, 1.82 mmol
)
in 4 mL CH
2
Cl
2
was added CF
3
CO
2
H (4
mL).
The solution was stirred at room temperature for 1
h. After removal of the solvent, the
resulting residue was dissolved in 4 mL 1N HCl and
extracted with 3
×
5 mL CH
2
Cl
2
.
The solvent (water) was removed by rotary evaporati
on and freeze drying. The
corresponding TFL·HCl salt was obtained in 87% yiel
d (352 mg). A 5 mL aqueous
solution of TFL·HCl (352 mg, 1.58 mmol) and NaOH (2
53 mg, 6.32 mmol) was cooled
to 0 °C. To this solution, Ac
2
O (224
μ
L, 2.37 mmol) was added dropwise. The mixture
was acidified to pH 2.5 with conc. HCl, and extract
ed with 3
×
100 ml EtOAc. The
combined organic layers were dried over MgSO
4
. Rotary evaporation of the solvent
yielded
6a
as a white solid (314 mg, 88%).
Compound
6a
(314 mg, 1.38 mmol) was subjected to enzymatic res
olution as previously
described. Compound
4b
was recovered from the reaction (190 mg). Compoun
d
4
was
obtained in 59% yield (71 mg).
Compound
3
was obtained from
6b
using the same procedures as above.
Deacetylation and Racemization of (2
R
,4
R
)-
N
-Acetyl-5,5,5-trifluoroleucine (3b)
3
Compound
3b
(113 mg, 0.49 mmol) was refluxed in 3N HCl for 3 h
ours. The solvent
was removed by rotary evaporation and lyophilizatio
n. The residue so obtained was
dissolved in 25 mL of acetic acid to which 0.05 eq
of benzaldehyde was added. The
reaction was stirred at 90 °C for 12 h. The solven
t was removed by rotary evaporation.
The residue was dissolved in 25 mL 1N HCl, then ext
racted with 3
×
20 mL EtOAc.
Removal of water delivered
2
in quantitative yield, and with C2 judged to be rac
emic by
1
H NMR.
Scheme S1
TFL (2S, 4S)
TFL (2R, 4R)
TFL (2S, 4R)
TFL (2R, 4S)
TFL (2S, 4S)
TFL (2R, 4R)
TFL (2R, 4S)
TFL (2S, 4R)
TFL (2S, 4S)
TFL (2R, 4R)
TFL (2R, 4S)
TFL (2S, 4R)
Enzyme
Enzyme
Column
Racemize position 2
S
R
N
H
Boc
O-
t
Bu
O
CF
3
N
H
Boc
O-
t
Bu
O
C
F
3
S
H
2
N
O
OH
CF
3
S
S
R
H
2
N
OH
O
CF
3
H
2
N
OH
O
CF
3
N
H
Ac
O
OH
CF
3
N
H
Ac
O
OH
CF
3
S
S
(2S, 4S)
(2S, 4R)
(2R, 4R)
(2R, 4S)
(2R, 4S)
(2R, 4R)
2
(TFL)
5a
5b
(a) Boc
2
O, NaHCO
3
, CH
3
OH, sonicate, 1 h; (b) Boc
2
O, DMAP,
t
BuOH
(anhyd); (c) flash column chromatography,
n
-pentane/MTBE (20:1); 52% over
3 steps from
2
; (d) CF
3
CO
2
H:CH
2
Cl
2
(1:1); (e) NaOH/H
2
O, Ac
2
O, 0 °C; (f)
Porcine kidney acylase I, pH 7.5, 25
°
C; 3N HCl; 54% over 3 steps from
5a
or
5b
; (g) 3N HCl, reflux; (h) CH
3
CO
2
H, 0.1 eq. C
6
H
5
CHO, 90 °C, 6 h; 95%
o
v
e
r
2
s
t
e
p
s
f
r
o
m
(
3b
+
4b
)
.
a, b, c
(2R, 4S)
(2R, 4R)
d, e
(2R, 4S)-
N
-Ac-TFL
(2R, 4R)-
N
-Ac-TFL
6a
6b
4
3
4b
3b
d, e
f
g, h
Table 1S.
Melting Transitions from CD
Protein
T
m
(
ο
οο
ο
C)
Leu-A1
55
SS-A1
65
SR-A1
65
SS-A1•SR-A1
[a]
68
[a]
This sample is an equimolar mixture of
SS-A1 and SR-A1. A1 made from the SS-
SR isomer mixture
2
exhibited a
T
m
of
67
o
C.
4
___________________________________________________
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Liebigs Ann. Chem.
1985
, 90-102.
2. Xing, X.; Fichera, A.; Kumar, K.
J. Org. Chem.
2002
,
67
, 1722-1725.
3. Yamada, S.; Hongo, C.; Yoshioka, R.; Chibata, I.
J. Org. Chem.
1983
,
48
, 843-846.
4. Tang, Y.; Ghirlanda, G.; Petka, W.A.; Nakajima,
T.; DeGrado, W. F.; Tirrell, D.A.
Angew. Chem. Int. Ed.
2001,
40
, 1494-1496