Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.
This journal is © The Royal Society of Chemistry 2016
Supporting Information for
Heat-enhanced peptide synthesis on Teflon-patterned paper
Frédérique Deissa,b*, Yang Yanga*, Wadim L. Matochkoa,c and Ratmir Derdaa†
Department of Chemistry and Alberta Glycomics Centre, University of Alberta, 11227 Saskatchewan
drive, Edmonton, AB T6G 2G2, Canada
a
b Present
address: Department of Chemistry and Chemical Biology, Indiana University-Purdue University,
Indianapolis, 402 N Blackford Street, Indianapolis, IN,46202, USA
c Present
address: Department of Antibody Engineering, Genentech Inc., 1 DNA Way, MS 433, South San
Francisco, CA, USA
* equal contribution
† corresponding author: E-mail: [email protected]
S1
Fig. S1: Heating set-up and distribution of temperature across the paper array.
Photograph of the frame holding four arrays at 20 cm distance over the infrared lamp (a). Heat
maps of the distribution of temperature throughout the 96 zones of the four arrays with each
pixel representing one zone (b-e, top-left to bottom-right). Numeric values indicate the
temperature in degree Celsius measured at these locations with an infrared thermometer. The
temperature in the zones without a measured value was interpolated from the neighboring zones
by Delaunay triangulation using the function TriScatteredInterp in Matlab®.
S2
Fig. S2: Absorbance measurement using a 96-quartz-well plate
(a) Photograph of the custom 96-quartz-well plate and three custom-made quartz cups used to
assemble this plate. (b) Due to the small variations in the thickness of the bottom of the custommade quartz cups, we observed a minor well-to-well variability in absorbance. These differences
were detectable only in dilute solutions. Example here represents well-to-well variability of the
“blank” 20% piperidine in DMF at 290 nm (150 µL per well) measured by SpectraMax M2e
plate reader (Molecular Devices). Once the variability in blank was measured, variability in the
subsequent absorbance measurements was negligible. Calibration curve established for Fmoc-Cl
between 0 and 6 mM (c) with a linear range below 0.6 mM of Fmoc-Cl (d) using the customemade 96-quart-cup plate. Average over 8 replicates; error bars represent two standard deviation.
S3
Fig. S3: Impact of flow-through on heat-enhanced reaction by using two types of paper
with different porosity. Evolution of the coupling of alanine or tryptophane over time between
2 and 24 minutes (a, c, e and g) and between 30 seconds and 6 minutes (b, d, f and h). The
experiments were performed independenely. Average over three replicates per conditions, error
bars represent two standard deviations as calculated from replicates within the same array.
S4
punches
map
row 1
row 2
row 3
row 4
row 5
row 6
row 7
row 8
col
col
col
col 1 col 2 col 3 col 4 col 5 col 6 col 7 col 8 col 9
10
11
12
βAla1 βAla1 βAla1 βAla1 βAla1 βAla1 βAla1 βAla1 βAla1 βAla1 βAla1 βAla1
βAla2 βAla2 βAla2 βAla2 βAla2 βAla2 βAla2 βAla2 βAla2 βAla2 βAla2 βAla2
A1
C1
Y1
R1
D1
S1
A1
C1
Y1
R1
D1
S1
A2
C2
Y2
R2
D2
S2
A2
C2
Y2
R2
D2
S2
A3
C3
Y3
R3
D3
S3
A3
C3
Y3
R3
D3
S3
A4
C4
Y4
R4
D4
S4
A4
C4
Y4
R4
D4
S4
A5
C5
Y5
R5
D5
S5
A5
C5
Y5
R5
D5
S5
A6
C6
Y6
R6
D6
S6
A6
C6
Y6
R6
D6
S6
Legend
β1
cellulose-βAla-Fmoc
β2
cellulose-βAla-βAla-Fmoc
A1
cellulose-βAla-βAla-Ala-Fmoc
R1
cellulose-βAla-βAla-Arg-Fmoc
A2
cellulose-βAla-βAla-Ala-Ala-Fmoc
R2
cellulose-βAla-βAla-Arg-Arg-Fmoc
A3
cellulose-βAla-βAla-Ala-Ala-Ala-Fmoc
R3
cellulose-βAla-βAla-Arg-Arg-Arg-Fmoc
A4
cellulose-βAla-βAla-Ala-Ala-Ala-Ala-Fmoc
R4
cellulose-βAla-βAla-Arg-Arg-Arg-Arg-Fmoc
A5
cellulose-βAla-βAla-Ala-Ala-Ala-Ala-Ala-Fmoc
R5
cellulose-βAla-βAla-Arg-Arg-Arg-Arg-Arg-Fmoc
A6
cellulose-βAla-βAla-Ala-Ala-Ala-Ala-Ala-Ala-Fmoc
R6
cellulose-βAla-βAla-Arg-Arg-Arg-Arg-Arg-Arg-Fmoc
C1
cellulose-βAla-βAla-Cys-Fmoc
D1
cellulose-βAla-βAla-Asp-Fmoc
C2
cellulose-βAla-βAla-Cys-Cys-Fmoc
D2
cellulose-βAla-βAla-Asp-Asp-Fmoc
C3
cellulose-βAla-βAla-Cys-Cys-Cys-Fmoc
D3
cellulose-βAla-βAla-Asp-Asp-Asp-Fmoc
C4
cellulose-βAla-βAla-Cys-Cys-Cys-Cys-Fmoc
D4
cellulose-βAla-βAla-Asp-Asp-Asp-Asp-Fmoc
C5
cellulose-βAla-βAla-Cys-Cys-Cys-Cys-Cys-Fmoc
D5
cellulose-βAla-βAla-Asp-Asp-Asp-Asp-Asp-Fmoc
C6
cellulose-βAla-βAla-Cys-Cys-Cys-Cys-Cys-Cys-Fmoc
D6
cellulose-βAla-βAla-Asp-Asp-Asp-Asp-Asp-Asp-Fmoc
Y1
cellulose-βAla-βAla-Tyr-Fmoc
S1
cellulose-βAla-βAla-Ser-Fmoc
Y2
cellulose-βAla-βAla-Tyr-Tyr-Fmoc
S2
cellulose-βAla-βAla-Ser-Ser-Fmoc
Y3
cellulose-βAla-βAla-Tyr-Tyr-Tyr-Fmoc
S3
cellulose-βAla-βAla-Ser-Ser-Ser-Fmoc
Y4
cellulose-βAla-βAla-Tyr-Tyr-Tyr-Tyr-Fmoc
S4
cellulose-βAla-βAla-Ser-Ser-Ser-Ser-Fmoc
Y5
cellulose-βAla-βAla-Tyr-Tyr-Tyr-Tyr-Tyr-Fmoc
S5
cellulose-βAla-βAla-Ser-Ser-Ser-Ser-Ser-Fmoc
cellulose-βAla-βAla-Tyr-Tyr-Tyr-Tyr-Tyr-Tyr-Fmoc
S6
cellulose-βAla-βAla-Ser-Ser-Ser-Ser-Ser-Ser-Fmoc
Y6
Fig. S4. Example of distribution of the synthesis of six homo-hexapeptide on a 96-zone paper
array, with each row displaying a truncation of the peptide sequence at another cycle to allow
quantifying the amount of Fmoc removed at each cycle.
S5
Fig. S5: Synthesis of homo-hexapeptides with 2-min coupling reaction. Conversion rates
calculated as the ratio Fmocn/Fmocester, averaged over four replicates (with the exception of A
and W averaged over 12 replicates). The values are reported in Table S1.
S6
IR
A*
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W*
Y
2nd βAla
64
76
76
66
66
75
75
66
75
66
75
51
51
75
76
76
51
51
64
76
1st
aa
55
50
59
47
54
69
28
43
40
49
57
32
45
25
29
64
35
28
38
64
2nd
aa
55
45
58
46
51
64
22
40
57
48
57
27
34
27
23
61
34
22
35
57
3rd aa
50
41
61
44
48
65
20
35
52
46
56
21
33
19
20
65
36
18
32
57
4th
aa
51
37
64
43
51
56
16
33
41
48
57
17
19
14
18
63
33
16
31
59
5th
aa
45
36
65
43
44
46
15
25
32
38
63
15
17
16
16
63
36
12
30
60
6th
aa
27
34
65
40
41
31
13
14
38
22
58
14
11
14
14
44
29
7
27
55
RT
A*
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W*
Y
2nd βAla
39
56
56
38
38
53
53
38
53
38
53
24
24
53
56
56
24
24
38
56
1st
aa
35
32
40
21
23
50
18
12
21
20
36
17
19
17
19
38
10
15
21
42
2nd
aa
34
29
39
21
22
46
13
9
26
18
40
12
12
13
12
38
13
13
20
40
3rd aa
34
27
38
20
20
40
9
7
18
16
37
7
7
8
7
38
13
11
18
37
4th
aa
31
22
41
17
19
13
6
7
20
17
38
5
6
4
4
35
11
9
14
38
5th
aa
25
18
37
18
17
14
5
5
15
15
41
3
4
4
5
31
12
7
13
37
6th aa
16
17
40
16
17
10
4
3
21
10
38
2
3
6
3
25
9
4
13
32
Table S1. Syntheses of homo-hexapeptide for 20 different amino acids; conversion in % of βAla
coupled to paper (Fmocn/Fmocester × 100), after each 2-minute coupling performed on top of the
infra-red lamp (labeled IR) compared to the control at room temperature (labeled RT). Each
value corresponds to the average of four replicates distributed over two individual arrays with the
exception of Ala and Trp noted with an asterisk (*) which were averaged over 12 replicates
distributed on six arrays.
S7
Table S2. Mass to charge ratio (m/z) and corresponding chemical structures of Ala2 to Ala6 with
their 2-βAla linker. All products were capped with acetic anhydride prior to LC-MS analysis.
S8
Fig. S6. Synthesis of homo-hexapeptides (a). Comparison of the average conversion of linker
to n-residue peptide when synthesizing Trp6 (b), Ala6 (c), and Arg6 (d), with heating or at room
temperature and coupling time of 3 or 10 minutes. The ratios Fmocn/ Fmocloading was calculated
from the amount of Fmoc loaded per unit area reported on Fig. 4.
S9
Fig. S7. Variability in the amount of Fmoc (mol/cm2) across four replicates within the same
array under 3-min IR coupling. 3-min IR coupling condition was chosen because we expect the
highest varibiality in synthesis upon short coupling conditions.
𝜎=
Deviation from the average is calculated as follow:
1
𝑁𝑖 𝑁𝑗
𝑖,𝑗
∑ (𝑎
1
𝑁𝑖
𝑖𝑗 ‒ 𝑎𝑖
𝑖
∑𝑎
𝑖
)2
1
𝑎𝑖 =
𝑁𝑗
,
𝑗
∑𝑎 ,
𝑖𝑗
where Ni and Nj represent total number of replicates and sample, j represents replicates and i
represents different coupling steps.
S10
Fig. S8. Amount of Fmoc (mol/cm2) loaded at the first -alanine coupling step across the entire
array under non-uniform (a) versus uniform (b) loading conditions. Under non-uniform
condition, we submerged the array in the activating solution without shaking; whereas under
uniform condition, the array was submerged in solution with mild rocking on a shaker.
S11
Fig. S9. Calibration curves used for extrapolating number of cells from grey-scale intensity. (a)
Calibration curve converting the number of MDA-MB-231-GFP cells to the corresponding greyscale intensity per peptide zone (A=0.16 cm2) measured by gel scanner. The curve was generated
by seeding a predefined number of cells inside paper-based array (b). After a brief incubation (15
minutes), the array was imaged by gel scanner.
S12
Fig. S10. Scheme for an aluminium grid insert used to hold peptide paper arrays submerged in a
Nunc Omni-Tray for cell adhesion assays.
S13