Supporting Information
for
Azide-Rich Peptides via an On-Resin Diazotransfer Reaction
Jeannette E. Marine, Xiaoli Liang, Shuang Song and Jonathan G. Rudick
Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, United States
ADDITIONAL SYNTHESIS SCHEMES .............................................................................................................. S1
EXPERIMENTAL SECTION ................................................................................................................................. S2
CHROMATOGRAMS AND MASS SPECTRA .................................................................................................... S6
REFERENCES ....................................................................................................................................................... S10
1
H AND 13C NMR SPECTRA..................................................................SERROR! BOOKMARK NOT DEFINED.
Additional Synthesis Schemes
Scheme S1. Synthesis of alkyne 6.
Scheme S2. Chemical structure of conjugate 7.
S1
Experimental Section
Assembly of Ac-YLKKLLKLLKKLLK-NH2 (3). Peptide 1a was assembled on ChemMatrix Rink amide resin
(0.50 mmol, 0.9628 g) in a glass-fritted peptide reaction vessel and cleaved from the resin to yield peptide 3. The
sequence of operations is detailed in the table below:
Table S1. Protocol for the synthesis of Ac-YLKKLLKLLKKLLK-NH2 (3).
Mass of Reagents (g)
Cycle
Fmoc-AA-OH
HBTU
1
Single Coupling: Fmoc-Lys(Boc)-OH
2.3219
1.8593
Capping
N-Fmoc Deprotection
2
Single Coupling: Fmoc-Leu-OH
1.7894
1.8655
Capping
N-Fmoc Deprotection
3
Single Coupling: Fmoc-Leu-OH
1.7750
1.8643
Capping
N-Fmoc Deprotection
4
Single Coupling: Fmoc-Lys(Boc)-OH
2.3715
1.8803
Capping
N-Fmoc Deprotection
5
Single Coupling: Fmoc-Lys(Boc)-OH
2.3225
1.8678
Capping
N-Fmoc Deprotection
6
Double Coupling: Fmoc-Leu-OH
1.7639
1.8588
1.7748
1.8564
Capping
N-Fmoc Deprotection
7
Single Coupling: Fmoc-Leu-OH
1.17690
1.8614
Capping
N-Fmoc Deprotection
8
Single Coupling: Fmoc-Lys(Boc)-OH
2.3292
1.8778
Capping
N-Fmoc Deprotection
9
Single Coupling: Fmoc-Leu-OH
1.7471
1.8877
Capping
N-Fmoc Deprotection
10
Single Coupling: Fmoc-Leu-OH
1.7781
1.8548
Capping
N-Fmoc Deprotection
11
Single Coupling: Fmoc-Lys(Boc)-OH
2.3251
1.8457
Capping
N-Fmoc Deprotection
12
Single Coupling: Fmoc-Lys(Boc)-OH
2.3099
1.8425
Capping
N-Fmoc Deprotection
13
Single Coupling: Fmoc-Leu-OH
1.7792
1.8679
Capping
N-Fmoc Deprotection
14
Single Coupling: Fmoc-Tyr(tBu)-OH
2.2917
1.8649

N -Fmoc Deprotection
Acetylation of the N-terminus
Cleavage and Side-Chain Deprotection
S2
HOBt
0.7898
0.7696
0.7660
0.7732
0.7898
0.7863
0.7522
0.7586
0.77700
0.7810
0.7963
0.7631
0.7760
0.7799
0.7877
Assembly of Ac-Y(tBu)LK(Boc)K(Alloc)LLK(Alloc)LLK(Boc)K(Alloc)LLK(Alloc)-NH-Resin (1b). Resinbound peptide 1b was assembled on ChemMatrix Rink amide resin (0.25 mmol, 0.5510 g) in a glass-fritted peptide
reaction vessel. The sequence of operations is detailed in the table below:
Table S2. Protocol for the synthesis of Ac-Y(tBu)LK(Boc)K(Alloc)LLK(Alloc)LLK(Boc)K(Alloc)LLK(Alloc)NH-resin (1b).
Cycle
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Mass of Reagents (g)
Fmoc-AA-OH
HBTU
1.1272
0.9184
Single Coupling: Fmoc-Lys(Alloc)-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Leu-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Leu-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Lys(Alloc)-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Lys(Boc)-OH
Capping
N-Fmoc Deprotection
Double Coupling: Fmoc-Leu-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Leu-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Lys(Alloc)-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Leu-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Leu-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Lys(Alloc)-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Lys(Boc)-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Leu-OH
Capping
N-Fmoc Deprotection
Single Coupling: Fmoc-Tyr(tBu)-OH
N-Fmoc Deprotection
Acetylation of the N-terminus
S3
HOBt
0.3845
0.8831
0.9281
0.3840
0.8853
0.9263
0.3827
1.1290
0.9239
0.3868
1.1739
0.9286
0.3876
0.8890
0.8904
0.9198
0.9174
0.3767
0.3862
0.8906
0.9201
0.3835
1.1157
0.9211
0.3878
0.8857
0.9207
0.3831
0.8835
0.9188
0.3879
1.1220
0.9287
0.3802
1.1192
0.9278
0.3811
0.8883
0.9250
0.3824
1.1482
0.9315
0.3844
(2,2-Dibutyl-5-methyl-1,3-dioxan-5-yl)methanol (S1). Compound S1 was prepared according to a previously
reported procedure.1 The reaction was performed in a 250-mL, one-neck flask equipped with a magnetic stir bar and
Soxhlet extractor containing molecular sieves. To an ice-water bath-cooled suspension of 1,1,1tris(hydroxymethyl)ethane (12.08 g, 0.1005 mol) in anhydrous C 6H6 (100 mL) and 5-nonanone (19.0 mL, 0.110
mol), BF3·Et2O (0.5 mL) was added slowly. The suspension was stirred for 30 min, and then heated at reflux for 7 d.
The reaction was cooled to room temperature, the residual solids were removed by filtration, and the crude product
was obtained by rotary evaporation of volatiles from the filtrate. The product was purified by flash column
chromatography (SiO2, hex/EtOAc 4:1 to 3:2) to give S1 as slightly yellow oil (16.16 g, 66%). TLC (SiO2, 7:3
hex/EtOAc): Rf = 0.37. 1H NMR (400 MHz, CDCl3, δ, ppm): 3.70 (d, J = 5.7 Hz, 2H), 3.64 (d, 2J = 11.7 Hz, 1H),
3.62 (d, 2J = 11.0 Hz, 1H), 3.58 (d, 2J = 11.7 Hz, 1H), 1.76 (m, 2H), 1.60 (m, 3H), 1.33 (m, 8H), 0.92 (t, J = 7.1 Hz,
3H), 0.90 (t, J = 7.0 Hz, 3H), 0.82 (s, 3H). 13C NMR (100 MHz, CDCl3, δ, ppm): 100.9, 66.6, 66.0, 36.9, 34.8, 30.1,
26.0, 25.3, 23.3, 23.2, 17.9. ESI–MS (m/z): [M + H]+ calcd for C14H28O3, 245.2; found 245.1. GPC: Mn = 190,
Mw/Mn = 1.06. The spectral data agree with literature.1
2,2-Bis[(2,2-dibutyl-5-methyl-1,3-dioxan-5-yl)methoxymethyl]ethene (S2). Compound S2 was prepared
according to a literature procedure.1 A solution of S1 (33.55 g, 137.3 mmol) in anhydrous THF (140 mL) was added
dropwise to an ice-water bath-cooled suspension of dry NaH (4.00 g, 166 mmol) in anhydrous THF (20 mL),
methallyl dichloride (6.3 mL, 54 mmol) and 15-crown-5 (2.7 mL, 14 mmol). The reaction mixture was heated at
reflux for 16 h under a N2 atmosphere while stirring. The reaction mixture was quenched with deionized water. The
product was extracted with EtOAc. The organic washings were combined, washed once with saturated NaCl (aq)
solution and dried over anhydrous MgSO4. The solids were removed by filtration. The volatiles were removed by
rotary evaporation. The resulting product was purified by flash chromatography (SiO 2, hex to 7:3 hex/EtOAc) to
yield S2 as a colorless liquid (28.41 g, 96%). TLC (SiO2, 7:3 hex/EtOAc): Rf = 0.67. 1H NMR (500 MHz, CDCl3, δ,
ppm): 5.13 (s, 2H), 3.97 (s, 4H), 3.66 (d, 2J = 11.7 Hz, 4H), 3.50 (d, 2J = 11.7 Hz, 4H), 3.39 (s, 4H), 1.72 (m, 2H),
1.60 (m, 2H), 1.32 (m, 16H), 0.90 (t, J = 6.9 Hz, 6H), 0.89 (t, J = 7.0 Hz, 6H), 0.85 (s, 6H). 13C NMR (125 MHz,
CDCl3, δ): 143.2, 113.3, 100.7, 73.4, 72.3, 66.1, 36.0, 34.3, 31.1, 25.8, 25.3, 23.3, 23.2, 18.6, 14.3. ESI–MS (m/z):
[M + H]+ calcd for C32H61O6, 541.4; found, 541.5. GPC: Mn = 480, Mw/Mn = 1.05. The spectral data agree with
literature.1
2,2-Bis[(2,2-dibutyl-5-methyl-1,3-dioxan-5-yl)methoxymethyl]ethanol (S3). Compound S3 was prepared
according to a literature procedure.1 To a solution of S2 (12.14 g, 22.45 mmol) in anhydrous THF (9 mL), a 0.5 M
9-BBN in THF solution (67 mL, 34 mmol) was added dropwise under a N 2 atmosphere. The reaction mixture was
stirred under a N2 atmosphere for 18 h. The reaction was cooled in an ice-water bath while 3 M NaOH (aq) solution
(30 mL, 90 mmol) was added dropwise to the reaction mixture. After the addition was complete, the reaction
mixture was stirred under a N2 atmosphere at room temperature for 15 min. The reaction vessel was cooled in an
ice-water bath and 30 wt% H2O2 (aq) solution (30 mL) was added dropwise to the reaction mixture. After the
addition was complete, the reaction mixture was stirred under a N 2 atmosphere at room temperature for 3 h. The
reaction mixture was saturated with K2CO3. The product was extracted with EtOAc. The organic washings were
combined and dried over anhydrous MgSO4. The solids were removed by filtration. The volatiles were removed
from the filtrate by rotary evaporation. The resulting product was purified by flash chromatography (SiO2, hex to 7:3
hex/EtOAc) to yield S3 as a colorless liquid (10.74 g, 86%). TLC (SiO 2, hex/EtOAc = 7:3): Rf = 0.42. 1H NMR
(500 MHz, CDCl3, δ): 3.75 (dd, J = 5.4 Hz, J = 5.5 Hz, 2H), 3.62 (dd, 2J = 11.7 Hz, 4J = 2.6 Hz, 4H), 3.57 (dd, 2J =
11.8 Hz, 3J = 6.3 Hz, 1H), 3.56 (dd, 2J = 9.3 Hz, 3J = 5.4 Hz, 1H), 3.54 (dd, 2J = 11.1 Hz, 3J = 5.4 Hz, 1H), 3.53 (dd,
2
J = 9.2 Hz, 3J = 6.2 Hz, 1H), 3.51 (d, 2J = 10.0 Hz, 4H), 3.44 (d, 2J = 8.9 Hz, 1H), 3.42 (d, 2J = 9.2 Hz, 2H), 3.40 (d,
2
J = 9.0 Hz, 1H), 2.57 (t, J = 5.7 Hz, 1H), 2.15 (m, 1H), 1.72 (m, 4H), 1.60 (m, 4H), 1.32 (m, 16H), 0.91 (t, J = 6.8
Hz, 6H), 0.90 (t, J = 7.0 Hz, 6H), 0.88 (s, 6H). 13C NMR (125 MHz, CDCl3, δ): 100.9, 74.7, 71.7, 66.1, 64.5, 41.6,
36.2, 34.4, 30.8, 25.9, 25.3, 23.3, 23.2, 18.5, 14.3. ESI–MS (m/z): [M + H]+ calcd for C32H63O7, 559.5; found, 559.5.
GPC: Mn = 620, Mw/Mn = 1.04. The spectral data agree with literature.1
2,2-Bis[(2,2-dibutyl-5-methyl-1,3-dioxan-5-yl)methoxy]ethyl propargyl ether (S4). To a solution of S3 (5.00 g,
8.95 mmol) in anhydrous THF (15 mL), 80 wt% propargyl bromide in toluene solution (1.5 mL, 13 mmol) and NaH
(0.30 g, 12 mmol) were added. The reaction was stirred at room temperature under N2 for 48 h. The reaction mixture
was quenched with deionized water. The product was extracted with EtOAc. The organic washings were combined,
washed once with saturated NaCl (aq) solution and dried over anhydrous MgSO4. The solids were removed by
filtration. The EtOAc was removed from the filtrate by rotary evaporation. The resulting product was purified by
flash column chromatography (SiO2, hex to 4:1 hex/EtOAc) to yield S4 as a colorless liquid (4.30 g, 81%). TLC
(SiO2, 4:1 hexanes/EtOAc): Rf = 0.63. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.10 (d, J = 2.4 Hz, 2H), 3.66 (d, J
S4
=11.7 Hz 4H), 3.56 (d, J = 5.9 Hz, 2H), 3.48 (d, J = 14.4 Hz, 4H), 3.46 (d, J = 8.7 Hz, 4H), 3.33 (dd, J1 = 8.0 Hz, J2
= 8.9 Hz, 4H), 2.39 (t, J = 2.4 Hz, 1H), 2.19 (m, 1H), 1.72 (m, 4H), 1.62 (m, 4H), 1.32 (m, 16H), 0.92 (t, J = 6.7 Hz,
6H), 0.90 (t, J = 6.9 Hz, 6H), 0.86 (s, 6H). 13C NMR (100 MHz, CDCl3, δ): 100.7, 80.2, 77.5, 74.3, 69.9, 68.9, 66.2,
58.6, 40.4, 35.6, 34.5, 31.5, 25.8, 25.4, 23.27, 23.25, 18.7, 14.3. HRMS–ESI (m/z): [M + H]+ calcd for C35H65O7,
597.4730; found, 597.4728. GPC: Mn = 620, Mw/Mn = 1.06.
2,2-Bis[2,2-di(hydroxymethyl)propyloxymethyl]ethyl propargyl ether (6). To a solution of S4 (0.50 g, 0.84
mmol) in a 1:1 v/v mixture of THF/MeOH (8 mL), Dowex 50WX2 (8.50 g) was added. The mixture was stirred at
room temperature under N2 for 2 d. The solids were removed by filtration. The resulting product was purified by
flash column chromatography (SiO2, CH2Cl2 to 19:1 CH2Cl2/MeOH) to yield S5 as colorless oil (0.2350 g, 81%).
TLC (SiO2, 19:1 CH2Cl2/MeOH): Rf = 0.18. 1H NMR (400 MHz, DMSO-d6, δ): 4.25 (t, J = 5.3 Hz, 4H), 4.09 (d, J =
2.4 Hz, 2H), 3.44 (d, J = 5.9 Hz, 2H), 3.37 (t, J = 2.4 Hz, 2H), 3.33 (d, J = 5.9 Hz, 4H), 3.24 (d, J = 5.3 Hz, 8H),
3.16 (t, J = 9.0 Hz, 4H), 2.03 (m, 1H), 0.76 (s, 6H). 13C NMR (100 MHz, DMSO-d6, δ): 80.4, 77.0, 73.4, 69.2, 68.0,
64.0, 57.7, 40.0, 35.6, 16.8. HRMS–ESI (m/z): [M + H]+ calcd for C17H33O7, 349.2226; found, 349.2218. GPC: Mn =
300, Mw/Mn = 1.08.
S5
Chromatograms and Mass Spectra
Figure S1. Chromatograms of peptide 3 (solid line) eluted with solvent A = H2O + 0.1% TFA and a) solvent B =
MeCN/H2O (9:1 v/v) + 0.1% TFA and b) solvent B = MeOH/H2O (9:1 v/v) + 0.1% TFA. The dashed line indicates
the solvent composition during elution of the peptide at 1 mL/min from a C18 column.
S6
Figure S2. Chromatogram of peptide 3 (solid line). The dashed line indicates the solvent composition during elution
of the peptide at 1 mL/min from a C4 column (solvent A = H 2O + 0.1% TFA; solvent B = iPrOH/MeCN/H2O (6:3:1
v/v/v) + 0.1% TFA).
Figure S3. MALDI-TOF Mass spectrum of peptide 3.
S7
Figure S4. Chromatograms of peptide 2a (solid line) eluted with solvent A = H2O + 0.1% TFA and a) solvent B =
i
PrOH/MeCN/H2O (6:3:1 v/v/v) + 0.1% TFA, and b) solvent B = iPrOH/MeCN/H2O (3:6:1 v/v/v) + 0.1% TFA. The
dashed line indicates the solvent composition during elution of the peptide at 1 mL/min from a C18 column.
Figure S5. MALDI-TOF Mass spectrum of peptide 2a.
S8
Figure S6. MALDI-TOF Mass spectrum of peptide 4.
Figure S7. Chromatograms of peptide 2b (solid line) eluted with solvent A = H2O + 0.1% TFA and a) solvent B =
MeCN/H2O (9:1 v/v) + 0.1% TFA, and b) solvent B = iPrOH/MeCN/H2O (3:6:1 v/v/v) + 0.1% TFA. The dashed
line indicates the solvent composition during elution of the peptide at 1 mL/min from a C18 column.
S9
Figure S8. MALDI-TOF Mass spectrum of peptide 2b.
Figure S9. Chromatogram of conjugate 7 (solid line) eluted with solvent A = H2O + 0.1% TFA and solvent B =
i
PrOH/MeCN/H2O (3:6:1 v/v/v) + 0.1% TFA. The dashed line indicates the solvent composition during elution of
the peptide at 1 mL/min from a C18 column.
References
(1)
Grayson, S. M.; Fréchet, J. M. J. J. Am. Chem. Soc. 2000, 122, 10335-1034
S10