Supporting Information
Kinetic study on the aromatic nucleophilic substitution
reaction of 3,6-dichloro-1,2,4,5-tetrazine by biothiols.
Daniela Andrade-Acuña1, José G. Santos1, William Tiznado2,
Álvaro Cañete1* and Margarita E. Aliaga1*
1
Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago
6094411, Chile.
2
Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andrés
Bello, Avenida República 275, Piso 3, Santiago, Chile.
* Corresponding authors. Fax: +56-(2)-26864744. E-mail address: acanetem@uc.cl (A.Cañete)
and mealiaga@uc.cl (M.E.Aliaga).
S1
Supporting Information
Table of contents.
Figure S1. Mass spectrum of 3,6-dichloro-1,2,4,5-tetrazine (DCT).
S3
Table S1. Experimental conditions and values of kobs1 (fast process) and kobs2
(slow process) for the reaction between DCT and biothiols (RSH) at different
pH values.
S3
Figure S2. Absorption spectra of DCT (200 M) in the presence of cysteine
(Cys, 10 equiv.) in aqueous solution (20 mM HEPES buffer, pH 7.4, 1%
DMSO), at different times. Insert shows the kinetic profile, at 370 nm, for
this reaction.
S4
Figure S3. Kinetic profile, at 370 nm for the reaction between DCT (200
M) in the presence of 1-propanethiol (1-PT, 100 equiv.) in aqueous solution
(20 mM HEPES buffer, pH 7.4, 1% DMSO).
S4
Figure S4. Plots of (A) kobs1 against thiolate concentration and (B) kobs2
against free amine concentration for the reaction of cysteine with DCT, in
aqueous solution (pH 7.4), at 25.0 °C, ionic strength 0.2 M (KCl).
S5
Table S2. Structures, atomic positions in Cartesian coordinates (Angstroms)
and computed relative energies (kcal·mol-1) of the two possible products from
the reaction of cysteine toward DCT.
S6
Table S3. Structures, atomic positions in Cartesian coordinates (Angstroms)
and computed relative energies (kcal·mol-1) of the two possible products from
the reaction of homocysteine toward DCT.
S7
Figure S5. Mass spectrum obtained after the addition of DCT to a solution
containing Hcy.
S8
Figure S6. Mass spectrum obtained after the addition of DCT to a solution
containing 2-ME.
S8
Figure S7. 1H-NMR spectrum of Cys (in D2O) after the addition of DCT.
S9
Synthesis of DCT-BA and DCT-NAC.
S9
Figure S8. 1H-NMR spectrum of DCT-BA.
S10
Figure S9. 1H-NMR spectrum of DCT-NAC in DMSO-d6.
S10
Figure S10. 1H-NMR spectrum of DCT-NAC in DMSO-d6.
S11
S2
Figure S1. Mass spectrum of 3,6-dichloro-1,2,4,5-tetrazine (DCT).
tetrazina_1_3 #4 RT: 0.33 AV: 1 NL: 1.06E7
T: + c EI Full ms [59.50-400.50]
61.031
100
95
90
85
87.094
80
75
70
Relative Abundance
65
60
55
50
45
40
35
63.048
150.132
30
89.095
25
152.131
20
15
10
5
64.059 71.194
0
60
70
82.051
80
90.097
90
98.086
100
110.078
117.059
110
120
126.113 130.110
130
154.129
155.133 163.112 169.386
141.338
140
150
160
170
m/z
Table S1. Experimental conditions and values of kobs1 (fast process) and kobs2 (slow
process) for the reaction between DCT and biothiols (RSH) at different pH values.a
Biothiols (RSH)
pH
XRS- b
103 [N]tot / M
102 kobs1 / s-1
103 kobs2 / s-1
Cysteine (Cys)
7.4
0.166
4.0-20.0
2.0-28.0
0.77-4.51
6.9
0.059
6.0-20.0
0.8-6.59
-
7.4
0.124
2.0-20.0
1.5-25.6
1.05-8.07
6.9
0.043
4.0-20.0
1.9-8.58
-
7.4
0.046
2.0-20.0
0.9-15.2
-
6.9
0.015
2.0-20.0
0.1-4.6
-
7.4
0.715
2.0-20.0
2.5-49.8
6.9-11.7
6.9
0.442
5.0-25.0
2.7-39.6
-
7.4
0.0049
5.0-25.0
0.1-2.2
-
6.9
0.0013
5.0-25.0
0.09-0.47
-
7.2
0.0156
2.0-20.0
2.2-9.1
-
6.9
0.0079
2.0-20.0
2.4-5.9
-
7.4
5.0×10-4
2.0-20.0
13.0-14.0
-
Homocysteine (Hcy)
Glutathione (GSH)
Cysteinyl-glycine (Cys-Gly)
2-mercaptoethanol (2-ME)
N-acetylcysteine (NAC)
1-propanethiol (1-PT)
a
In HEPES buffer, at 25.0 ± 0.1 °C, and an ionic strength of 0.2 M (maintained with KCl).
b
XRS- is the molar fraction of thiolate ions.
S3
Figure S2. Absorption spectra of DCT (200 M) in the presence of cysteine (Cys, 10
equiv.) in aqueous solution (20 mM HEPES buffer, pH 7.4, 1% DMSO), at different
times.
Insert
shows
the
profile,
at
370
nm,
300
400
500
for
this
reaction.
0.6
Absorbance (AU)
2.0
Absorbance (AU)
kinetic
1.5
1.0
0.5
0.4
0.3
0.2
0
200
100
0.5
Time (s)
0.0
200
500
400
300
600
Wavelength (nm)
Figure S3. Kinetic profile, at 370 nm for the reaction between DCT (200 M) in the
presence of 1-propanethiol (1-PT, 100 equiv.) in aqueous solution (20 mM HEPES
buffer, pH 7.4, 1% DMSO).
Absorbance (AU)
0.20
0.15
0.10
0.05
0.00
0
100
200
300
400
500
Time (s)
S4
Figure S4. Plots of (A) kobs1 against thiolate concentration and (B) kobs2 against free amine
concentration for the reaction of cysteine with DCT, in aqueous solution (pH 7.4), at 25.0 °C,
ionic strength 0.2 M (KCl).
0.005
0.35
0.21
0.14
0.07
0.00
0.0
(B)
0.004
0.28
kobs2 / s-1
kobs1 / s-1
(A)
0.003
0.002
0.001
0.1
0.2
0.3
102 [RS-]/M
0.4
0.000
0
1
2
3
4
5
105 [N]/M
S5
Table S2. Structures, atomic positions in Cartesian coordinates (Angstroms) and computed
relative energies (kcal·mol-1) of the two possible products from the reaction of cysteine toward
DCT. The calculations were performed at PBE0/6-31+G* level.
Structure
Atomic numbers and Cartesian coordinates (Å)
7
6
6
7
7
7
16
16
6
1
1
6
1
1
6
1
6
1
6
8
8
1
6
8
8
1
7
1
7
1
1
1
1.174354000
1.142082000
-1.082892000
0.039555000
0.036120000
-1.094241000
3.907701000
-4.136488000
3.886314000
4.882238000
3.170762000
-4.223722000
-5.296300000
-3.830648000
-3.554297000
-4.191346000
3.603020000
4.331939000
3.885081000
4.664988000
3.232822000
3.491533000
-3.565837000
-2.587560000
-4.824153000
-4.781347000
2.304273000
2.251360000
-2.250841000
-2.075324000
-2.795011000
2.610907000
-0.475606000 -0.846563000
-0.307221000 0.485949000
-0.656023000 -0.723975000
-0.644055000 -1.463249000
-0.432066000 1.236566000
-0.620302000 0.615850000
2.685806000 0.420293000
-0.410294000 1.816345000
1.084827000 -0.433423000
0.957409000 -0.864807000
1.093551000 -1.258763000
-1.115674000 0.143411000
-1.174392000 -0.061179000
-2.136048000 0.137227000
-0.343029000 -1.021387000
-0.520346000 -1.896306000
-0.088332000 0.507742000
-0.035800000 1.326864000
-1.416161000 -0.198733000
-1.544667000 -1.110324000
-2.444466000 0.367335000
-3.244617000 -0.121908000
1.165557000 -0.792520000
1.863530000 -0.690009000
1.627891000 -0.759532000
2.576670000 -0.547863000
-0.021744000 1.135575000
-0.110230000 2.140031000
-0.831056000 -1.420390000
-0.775835000 -2.415931000
-0.508635000 1.925368000
2.681923000 0.784400000
7
6
6
7
7
7
16
16
6
1
1
6
1
1
6
1
6
1
6
8
8
1
6
8
8
1
7
1
7
1
1
1
1.174405000
1.142045000
-1.082871000
0.039643000
0.036064000
-1.094280000
3.908557000
-4.136611000
3.886676000
4.882573000
3.171148000
-4.223721000
-5.296285000
-3.830557000
-3.554309000
-4.191278000
3.602997000
4.331839000
3.884812000
4.664809000
3.232194000
3.490776000
-3.566048000
-2.587872000
-4.824433000
-4.781742000
2.304182000
2.251203000
-2.250780000
-2.075288000
-2.795095000
2.611860000
-0.474946000 -0.846902000
-0.307097000 0.485669000
-0.655134000 -0.724569000
-0.643009000 -1.463759000
-0.432432000 1.236159000
-0.620264000 0.615281000
2.685411000 0.420756000
-0.412240000 1.815880000
1.084558000 -0.433187000
0.956885000 -0.864558000
1.093620000 -1.258543000
-1.115914000 0.142221000
-1.174517000 -0.062475000
-2.136248000 0.135001000
-0.342004000 -1.021743000
-0.518484000 -1.896888000
-0.088638000 0.507803000
-0.036360000 1.327010000
-1.416461000 -0.198778000
-1.545108000 -1.110270000
-2.444646000 0.367110000
-3.244826000 -0.122156000
1.166357000 -0.791362000
1.864378000 -0.688254000
1.628465000 -0.757813000
2.577039000 -0.545197000
-0.021754000 1.135516000
-0.111265000 2.139886000
-0.829442000 -1.421157000
-0.773733000 -2.416666000
-0.510298000 1.924719000
2.681663000 0.785208000
G
0.0
8.0
S6
Table S3. Structures, atomic positions in Cartesian coordinates (Angstroms) and computed
relative energies (kcal·mol-1) of the two possible products from the reaction of Homocysteine
toward DCT. The calculations were performed at PBE0/6-31+G* level.
Structure
Atomic numbers and Cartesian
coordinates (Å)
6
6
7
7
7
7
16
16
6
6
1
1
6
1
1
6
1
6
6
1
1
6
1
1
6
1
7
1
8
8
1
7
1
8
8
1
1
1
-1.244092000
1.249356000
0.861602000
-0.411132000
0.417891000
-0.854941000
-6.857852000
6.926113000
5.208607000
4.966634000
5.058113000
4.512819000
3.535537000
5.655333000
5.165708000
3.441738000
3.327817000
-5.166305000
-4.964142000
-4.445723000
-5.014147000
-3.540545000
-5.175059000
-5.671929000
-3.492168000
-3.310359000
2.583452000
2.765814000
4.046472000
2.638108000
2.650629000
-2.579966000
-2.764967000
-4.086801000
-2.744578000
-2.785618000
-6.807441000
6.835180000
-0.069236000
0.010772000
0.426189000
0.379237000
-0.450653000
-0.497312000
1.775676000
-1.585251000
-1.142751000
0.308604000
-1.267754000
-1.801461000
0.759170000
0.967058000
0.444518000
2.275933000
0.568966000
1.161458000
-0.282705000
1.830441000
1.249382000
-0.781314000
-0.383381000
-0.932901000
-2.296443000
-0.612338000
0.011906000
-0.058460000
3.056719000
2.652455000
3.625231000
-0.049235000
0.021429000
-3.073574000
-2.676880000
-3.648380000
1.770186000
-2.890945000
0.289384000
-0.254430000
0.963036000
1.242998000
-1.203057000
-0.923024000
-0.081582000
-0.132443000
0.304637000
-0.087249000
1.382303000
-0.222227000
0.230128000
0.452609000
-1.159833000
0.071150000
1.288808000
-0.360115000
0.079096000
0.118746000
-1.441936000
-0.207374000
1.153777000
-0.444027000
-0.024149000
-1.264921000
-0.554101000
-1.548221000
0.765189000
-0.937968000
-0.960579000
0.581665000
1.575188000
-0.730846000
1.026697000
1.064433000
1.264908000
0.181711000
6
6
7
7
7
7
16
16
6
6
1
1
6
1
1
6
1
6
6
1
1
6
1
1
6
1
7
1
-1.155544000
1.149692000
1.096093000
-0.085252000
0.079964000
-1.101190000
-2.699763000
2.691623000
3.843287000
5.258544000
3.598019000
3.734034000
6.297188000
5.381701000
5.458723000
7.648938000
6.054710000
-3.848259000
-5.269941000
-3.660500000
-3.674874000
-6.295287000
-5.404414000
-5.473302000
-7.680088000
-6.123469000
6.236112000
6.812088000
0.556269000
-0.469684000
0.769690000
1.294419000
-1.198869000
-0.673394000
1.316557000
-1.251194000
0.041331000
-0.514270000
0.315228000
0.930856000
0.565543000
-1.363764000
-0.879433000
-0.121504000
0.964657000
0.010609000
0.506989000
-0.902567000
-0.206656000
-0.587165000
0.800250000
1.391591000
0.052641000
-0.899958000
1.651699000
2.433545000
0.102035000
-0.266991000
0.248177000
0.437574000
-0.623589000
-0.436041000
0.403443000
-0.521659000
0.039288000
-0.054524000
1.070009000
-0.585132000
0.294139000
0.627160000
-1.071707000
0.456665000
1.286339000
-0.132323000
0.096964000
0.436627000
-1.190917000
-0.246567000
1.147675000
-0.517461000
-0.244161000
-1.283725000
-0.662283000
-0.365513000
G
0.0
14.2
S7
1
8
8
1
7
1
1
8
8
1
6.587692000
8.006537000
8.410211000
9.227585000
-6.112872000
-6.395966000
-6.684637000
-8.120763000
-8.367818000
-9.213647000
1.351991000
-0.706226000
-0.041764000
-0.537457000
-1.738523000
-1.523110000
-2.519137000
0.735771000
-0.195465000
0.279925000
-1.567849000
1.452335000
-0.653199000
-0.471970000
0.612744000
1.565076000
0.304892000
-1.138790000
0.888344000
0.815215000
Figure S5. Mass spectrum obtained after the addition of DCT to a solution containing Hcy.
Figure S6. Mass spectrum obtained after the addition of DCT to a solution containing
2-ME.
rx_nn_1 #78 RT: 5.63 AV: 1 SB: 56 0.88-4.84 NL: 7.29E5
T: + c EI Full ms [ 59.50-400.50]
105.18
100
90
80
Relative Abundance
70
60
150.17
50
40
30
137.18
20 82.17
110.13
98.20
83.18
84.15 96.11
10
118.15
116.20
233.35
172.18
152.16
128.12
119.15
121.17
219.32
134.18
149.20
157.23 164.20
174.18
232.32 235.34
185.29 190.18
198.23
209.33
239.50
244.41
221.32
0
80
90
100
110
120
130
140
150
170
160
180
190
200
210
220
230
240
m/z
S8
Figure S7. 1H-NMR spectrum of Cys (in D2O) after the addition of DCT in DMSO-d6.
Synthesis of DCT-BA
DCT (200 mg, 1.32 mmol) was dissolved in acetonitrile (5 mL), then N-acetylcysteine
(314 µL, 3.18 mmol), was added under nitrogen atmosphere. After stirring at 80 °C for
24 h, the mixture was washed with acetonitrile/diethyl ether (1:1) (20 mL × 3). The
organic layer was dried was carefully evaporated under reduced pressure. The red
residue obtained was purified by multiple washed with acetonitrile/diethyl ether (1:5),
due to the unstability of compound DCT-BA (196 mg, 58%).
H NMR (400 MHz, DMSO-d6) δ: 8.82 (s, 1H), 3.34 (d, 2H, J = 31.4 Hz), 1.57 (m,
2H), 1.36 (m, 2H), 0.90 (t, 3H).
1
Synthesis of DCT-NAC
DCT (200 mg, 1.32 mmol) was dissolved in acetonitrile (5 mL), then N-acetylcysteine
(519 mg, 3.18 mmol), was added under nitrogen atmosphere. After stirring at 80 °C for
24 h, the mixture was washed with acetonitrile/diethyl ether (1:1) (20 mL × 3). The
organic layer was dried was carefully evaporated under reduced pressure. The red
residue obtained was purified by multiple washed with acetonitrile/diethyl ether (1:5),
due to the unstability of compound DCT-NAC (260 mg, 49%).
H NMR (400 MHz, CDCl3) δ: 8.32 (s, 1H), 4.42 (s, 1H), 2.97 (d, 2H, J = 73.5 Hz),
1.82 (s, 3H).
1
S9
Figure S8. 1H-NMR spectrum of DCT-BA in DMSO-d6.
Figure S9. 1H-NMR spectrum of NAC in DMSO-d6.
a
a
S10
Figure S10. 1H-NMR spectrum of DCT-NAC in DMSO-d6.
S11