Improved
S
ynthesis of 4
-
C
yanotryptophan and
O
ther
T
ryptophan
A
nalog
ue
s in
A
queous
S
olvent
U
sing
V
ariants of TrpB from
Thermotoga maritima
Christina E. Boville
, David K. Romney, Patrick J. Almhjell, Michaela Sieben, Frances H. Arnold*
Division of Chemistry and Chemical Engineering 210
-
41, California Institute of Technology, 1200
East California Boulevard, Pasadena, California 91125, United States
*E
-
mail:
france
s@cheme.caltech.edu
, Phone
:
(626) 395
-
4162, Fax: (626) 568
-
8743
S
1
Table of Contents
Results of site
-
saturation mutagenesis libraries
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S
2
Figure S1.
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S
2
Figure S2.
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S
3
Figure S3.
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S
4
LCMS calibration curves for Chart 1
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...........
S
5
Figure S4.
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S
5
Table S1. HPLC data for Figure 2
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S
6
Table S2. HPLC data for reactions after 1 hour
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S
6
Table S3. HPLC data for Chart 1 and indole
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S
7
Table S4. Primers for construction of recombination libraries
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................................
....
S
8
Table S5. Primers for construction of site
-
saturation libraries
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....
S
8
4
-
cyanotryptophan NMR spectrum for Scheme 2
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S
9
References
................................
................................
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S
9
S
2
Results of
screening
site
-
saturation
mutagenesis
libraries
Figure S1.
Site
-
saturation
mutagenesis
of
residue
30 in
Tm9D8
.
Following
the procedure in the
Experimental Section
,
80 colonies (4
-
fold oversampling) were assayed for 4
-
CN
-
Trp production at 50 °C.
Although many variants showed parent
-
like activity,
the top two variants were found to be parent (G30)
.
S
3
Figure S2.
S
ite
-
saturation
mutagenesis
of residue 228 in
Tm
9D8.
Following
the procedure in the
Experimental Section,
80 colonies (4
-
fold oversampling) were assayed for 4
-
CN
-
Trp production at 50 °C.
The top two variants were identified as parent (S228)
.
S
4
Figure S3.
Site
-
s
aturation
mutagenesis
of residue 184 in Tm9D8.
Following
the procedure in the
Experimental Section, 80 colonies (4
-
fold oversampling) were assayed for 4
-
CN
-
Trp production at 50 °C.
I184L was found in two of the top variants, but this mutation was not as be
neficial as I184F in the rescreen.
S
5
LCMS calibration curves for Chart 1
Figure S4.
Using an authentic product standard, mixtures of starting material and product at different ratios
(9:1, 3:1, 1:1, 1:3, and 1:9) were used to
generate
a calibration curve. Each mixture was prepared in
duplicate with a final concentration
o
f 1 mM in 1:1 1
-
M aq. HCl/ CH
3
CN. Mixtures were analyzed with LCMS
at 254 nm and 280 nm (reference 360 nm, bandwidth 100 nm) and 330 nm (no reference wavelength) and
w
ere correlated to the actual ratios by a linear relationship. The correlation for 4
-
nitrotryptophan was
published previously.
1
For tryptophan and 5
-
chloro
-
7
-
iodotryptophan, the HPLC yield was approximated by
comparing absorption peaks of product and starting material at 280 nm.
y = 1.4639x + 0.0107
R² = 0.9824
y = 3.6887x
–
0.0394
R² = 0.9992
0
10
20
30
40
0
2
4
6
8
10
observed ratio
actual ratio of 4
-
cyanoindole to 4
-
cyanotryptophan
254 nm
280 nm
y = 1.465x
–
0.0198
R² = 0.9999
y = 1.255x
–
0.0298
R² = 0.9992
0
5
10
15
0
2
4
6
8
10
observed ratio
actual ratio of 4
-
bromoindole to 4
-
bromotryptophan
254 nm
280 nm
y = 1.2337x + 0.1514
R² = 0.9992
y = 1.0951x + 0.0254
R² = 0.9999
y = 0.6793x + 0.0367
R² = 0.9998
0
2
4
6
8
10
12
0
2
4
6
8
10
observed ratio
actual ratio of 5
-
nitroindole to 5
-
nitrotryptophan
254 nm
330 nm
280 nm
y = 0.7454x
–
0.0149
R² = 0.9998
y = 0.5835x
–
0.0083
R² = 0.9996
0
2
4
6
8
0
2
4
6
8
10
observed ratio
actual ratio of 5
-
Br
-
7
-
F
-
indole to 5
-
Br
-
7
-
F
-
tryptophan
254 nm
280 nm
S
6
Table
S1. HPLC data for Figure 2
Temperature
(
°
C)
37
37
50
50
75
75
Tm
2F3
254 nm
:
5.68%
5.76%
17.1%
17.3%
15.1%
18.7%
280 nm
:
3.19%
3.00%
10.8%
10.0%
7.44%
9.78%
Tm
9D8
254 nm
:
30.5%
28.7%
38.0%
36.2%
18.0%
16.5%
280 nm
:
16.6%
15.2%
21.9%
19.5%
11.7%
10.8%
Tm
9D8*
254 nm
:
69.2%
66.5%
70.1%
71.1%
35.1%
35.9%
280 nm
:
48.1%
47.6%
49.9%
48.7%
19.3%
20.5%
Temperature (
°
C)
37
37
50
50
75
75
Tm
2F3
Corrected
254 nm:
8.11%
8.22%
23.2
%
23.5%
20.6
%
25.3
%
280 nm:
10.8
%
10.2
%
30.7
%
29.0
%
22.8
%
28.5
%
Tm
9D8
Corrected
254 nm:
39.2%
37.2%
47.5%
45.5%
24.4%
22.5%
280 nm:
42.1%
39.6%
50.5%
46.9%
32.7%
30.7%
Tm
9D8*
Corrected
254 nm:
77.1%
74.8%
77.9%
78.7%
44.4%
45.1%
280 nm:
76.7%
76.4%
77.9%
77.1%
46.7%
48.6%
Table S
2
. HPLC data for
reactions after 1 hour
a
HPLC yield
Corrected
Catalyst
2
54
nm
2
54
nm
254 nm
#1
#2
#3
Average
Std. Dev.
Tm
2F3
b
0.834
%
0.800%
0.840%
1.22%
1.17%
1.22%
1.20%
0.03%
Tm
9D8
b
3.7%
3.7%
4.3%
5.4%
5.3%
6.1%
5.6%
0.4%
Tm
9D8*
c
3.9%
3.8%
4.3%
5.6%
5.5%
6.1%
5.7%
0.3%
a
Maximum 1
000 turnovers.
b
Reactions conducted at 50 °C.
c
Reactions conducted at 37 °C.
S
7
Table S
3
.
HPLC data for Chart 1 and indole
4
-
bromotryptophan:
HPLC yield at indicated wavelength
Catalyst
254 nm
254 nm
Average
Corrected
280 nm
280 nm
Average
Corrected
Tm
2F3
58.3%
58.1%
58.2%
66.5%
60.9%
59.7%
60.3%
64.6%
Tm
9D8
63.2%
65.7%
64.4%
71.9%
67.1%
68.0%
67.6%
71.1%
Tm
9D8*
69.5%
68.6%
69.1%
75.8%
72.7%
72.2%
72.4%
75.3%
4
-
nitrotryptophan:
HPLC yield at indicated wavelength
Catalyst
254 nm
254 nm
Average
Corrected
330 nm
330 nm
Average
Corrected
Tm
2F3
3.45%
2.51%
2.98%
2.52%
1.09%
1.14%
1.11%
2.58%
Tm
9D8
1.74%
2.70%
2.22%
1.87%
1.10%
1.08%
1.09%
2.53%
Tm
9D8*
–
–
–
–
–
–
–
–
5
-
nitrotryptophan:
HPLC yield at indicated
wavelength
Catalyst
254 nm
254 nm
Average
Corrected
330 nm
330 nm
Average
Corrected
Tm
2F3
16.4%
17.9%
17.2%
20.9%
19.3%
19.4%
19.3%
20.9%
Tm
9D8
4.28%
4.97%
4.62%
5.68%
4.79%
4.92%
4.86%
5.3%
Tm
9D8*
5.57%
5.80%
5.68%
6.98%
6.77%
6.97%
6.87%
7.5%
5
-
bromo
-
7
-
fluorotryptophan
:
HPLC yield at indicated wavelength
Catalyst
254 nm
254 nm
Average
Corrected
280 nm
280 nm
Average
Corrected
Tm
2F3
40.8%
42.1%
41.4%
34.3%
46.8%
48.1%
47.4%
34.3%
Tm
9D8
96.8%
97.1%
96.9%
94.1%
97.1%
97.7%
97.4%
94.4%
Tm
9D8*
94.4%
95.4%
94.9%
91.5%
95.1%
96.4%
95.7%
91.7%
5
-
chloro
-
7
-
iodotryptophan
:
tryptophan:
HPLC yield
HPLC
yield
Catalyst
280 nm
280 nm
Average
Catalyst
280 nm
280 nm
Average
Tm
2F3
2.3%
2.2%
2.3%
Tm
2F3
99.3%
97.3%
98.3%
Tm
9D8
19.7%
20.6%
20.2%
Tm
9D8
90.6%
88.3%
89.4%
Tm
9D8*
18.9%
17.8%
18.4%
Tm
9D8*
79.0%
79.7%
79.3%
S
8
Table S
4
. Primers for construction of recombination libraries
Fragment
Forward Primer
Reverse Primer
Fragment 1:
Nde
I to E30
GAAATAATTTTGTTTAACTTTAAGA
AGGAGATATACATATG
CTCATCTTTCATGATTYCTTCGTA
CGCAGCTTC
Fragment 2:
E30 to I184
GAAGCTGCGTACGAAGRAATCAT
GAAAGATGAG
ACCAACCACAGAGCCGTWCACGT
AATAGGTGGT
Fragment 3:
I184 to G228
ACCACCTATTACGTGWTCGGCTC
TGTGGTTGGT
AGCGTTAGAACCACCGCYCACGC
ACGCAACGAT
Fragment 4:
G228 to
Xho
I
ATCGTTGCGTGCGTGRGCGGTGG
TTCTAACGCT
GCCGGATCTCAGTGGTGGTGGTG
GTGGTGCTCGAG
Gene
Assembly
GAAATAATTTTGTTTAACTTTAAGA
AGGAGATATACATATG
GCCGGATCTCAGTGGTGGTGGTG
GTGGTGCTCGAG
Table S
5
. Primers for construction of site
-
saturation libraries
t
arget
Tm
9D8
r
esidue
f
orward
primer
r
everse
p
rimer
E30
TGCGTACGAA
XXX
ATCATGAAAGATGAG
GCTTCCAGTTCTTCCAGAG
I184
CTATTACGTG
XXX
GGCTCTGTGGTTGG
GTGGTCTGCAGGTTGGTA
G228
TGCGTGCGTG
XXX
GGTGGTTCTA
ACGATGTAGTCCGGCAGAC
The codon indicated as XXX is replaced with NDT, VHG, or TGG.
S
9
4
-
cyanotryptophan NMR spectrum for Scheme 2
References
(1) Romney, D. K.; Murciano
-
Calles, J.; Wehrmüller, J. E.; Arnold, F. H. Unlocking Reactiv
ity of TrpB: A
General Biocatalytic Platform for Synthesis of Tryptophan Analogues.
J. Am. Chem. Soc.
2017
,
139
(31), 10769
–
10776.