Neutral peptide biradicals formed by dissociative electron transfer

Article abstract of DOI:10.1021/ol0160452

(Matrix presented) A new method for the generation of transient neutral biradicals in liquid solution is reported. Photoinduced electron transfer in aqueous solution of the structures shown above leads to neutral biradicals with peptide spacers. Exchange

Full text of DOI:10.1021/ol0160452

Supporting Information for:
Neutral Peptide Biradicals Formed by Dissociative Electron
Transfer
Colin S. Burns, Lakecia Rochelle, and Malcolm D. E. Forbes*
Venable and Kenan Laboratories
Department of Chemistry, CB #3290
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina 27599
________________________________________________________________________
*to whom correspondence should be addressed. Email: mdef@unc.edu.

Experimental Section
TREPR Methods. The time-resolved experiments were performed at X-band
using a JES-RE1X JEOL, USA, Inc. X-Band ESR spectrometer equipped for direct
detection using a boxcar integrator as described in previous publications.1,2 All systems
studied were flash photolyzed at 308 nm by an eximer laser (Lambda Physik LPX 110I)
operating at a repetition rate of 60 Hz. Sample solutions were bubbled with nitrogen
prior to and during photolysis, and were continuously circulated through a 0.5 mm path
length quartz flat cell centered in the microwave cavity. Sample concentrations ranged
from 0.017 - 0.096 M in peptide (i.e. compounds n = 0-4).
Simulations. Spectral EPR simulations of biradicals were performed using our
standard spin-correlated radical pair (SCRP) simulation program. The model used has
been described in previous publications.3 The hyperfine couplings for the tyrosyl radical
(Figure 1A) are: H(benzylic) = 7.5 G, H(ortho) = 6.1 G, H(meta) = 1.2 G, H(Cα) = 0.4 G
and the g-factor is 2.0041. The solvated electron has a g-factor of 2.0002, but has no
hyperfine couplings. The simulation parameters for the phenyl radical (Figure 1B) are:
H(ortho) = 17.0 G and H(meta) = 6.0 G, and the g-factor is 2.0032. In this case the
couplings for the tyrosyl radical are the same as listed for Figure 1A. The hyperfine
couplings and g-factors used for simulation of the biradicals are the same as used in the
monoradical simulations and J = -1600 MHz for n = 2 and –1100 MHz for n = 3. The
excitation wavelength in all cases is 308 nm.
Solvents and Materials. All reagents and solvents were obtained from the
Aldrich chemical company and used without further purification with the following
exceptions: The water used for all experiments was obtained from a Millipore Milli-
Qplus filtration system (conductivity 18.2 mΩ cm-1), methanol (Burdick and Jackson,
99.9+%), dichloromethane (Mallinckrodt) was distilled from calcium hydride,
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP)

(Sigma), glycine methyl ester hydrochloride (Gly-Ome•HCl) (Sigma), sodium hydroxide
(Mallinckrodt, 98.7%).
(4-bromobenzyl-1-carbonyl)-tyrosine methyl ester (Bba-Tyr-OMe) (6). To a
dry 500 mL three-neck roundbottom flask with a stir bar was added 8.3709 g (43 mmol)
tyrosine methyl ester, 9.4907 g (47 mmol) 4-bromobenzoic acid, 20.8823 g (47 mmol)
BOP, 6.3799 g (47 mmol) 1-hydroxybenzotriazole (HOBt), 50 mL dry
dimethylformamide (DMF), and 14.95 mL (86 mmol) diisopropylethylamine (DIEA).
The solution was left to stir overnight under nitrogen. The DMF was removed using
vacuum and gentle heat to obtain a yellow oil. The oil was dissolved in ethyl acetate
(EtOAc) and washed with 10% HCl solution, saturated NaHCO3 solution, and then brine
solution. The organic layer was dried with MgSO4 and then filtered. The volume of
solvent was reduced with heating and the flask left to sit out open to the air overnight.
Obtained four crops of white crystals, 6, having a combine mass of 14.4884 g (89.3%
1
yield). H NMR (acetone-d6) 8.20 (singlet, 1H), 7.90 (broad doublet, 1H), 7.75 (doublet,
2H), 7.62 (doublet, 2H), 7.10 (doublet, 2H), 6.64 (doublet, 2H), 4.70 (multiplet, 1H), 3.68
(singlet, 3H), 3.08 (multiplet, 2H).
(4-bromobenzyl-1-carbonyl)-tyrosine (Bba-Tyr) (1). Placed 2.4005 g (6.3
mmol) of 6 into a 500 mL roundbottom flask. Added 200 mL 7:3 MeOH/H2O and
0.8033 g (19 mmol) LiOH•H2O. The solution was left to stir overnight open to the
atmosphere. Removed MeOH under reduced pressure with heating and a white solid
precipitated out of solution. Acidified solution by adding HCl and cooled in an ice bath.
Filtered white solid and rinsed it with 10% HCl and then with deionized (DI) H2O. The
1
product was dried in a dessicator. Obtained 2.3115 g (95.0% yield) of 1. H NMR (200
MHz, acetone-d6) 7.84 (multiplet, 1H), 7.78 (doublet, J = 8 Hz, 2H), 7.62 (doublet, J = 8
Hz, 2H), 7.15 (doublet, J = 8 Hz, 2H), 6.73 (doublet, J = 8 Hz, 2H), 4.84 (multiplet, 1H),
3.14 (multiplet, 2H); 13C NMR (300 MHz, acetone-d6) 172.3, 166.0, 156.1, 133.8, 131.4,
130.3, 129.2, 128.1, 115.1, 54.4, 36.0; HRMS (FAB, MeOH) m/z = 364.0203 (calc. m/z

= 364.0184 for C16H14BrNO4). UV/Vis (0.1001M NaOH) λmax = 241.1 nm, ε241.1 = 24,000;
λmax = 293.0 nm, ε293.0 = 2200; ε308.0 = 1000.
(4-bromobenzyl-1-carbonyl)-glycine methyl ester (Bba-Gly-OMe) (7). In a
500 mL three-neck flask containing a stir bar was placed 8.37 g (42 mmol) 4-
bromobenzoic acid, 5.72 g (46 mmol) Gly-OMe•HCl, 50 mL dry CH2Cl2, 22 mL (126
mmol) DIEA, and 1.23 g (10 mmol) 4-dimethylaminopyridine (DMAP) as a catalyst. A
yellow oil was obtained. The oil was dissolved in MeOH and H2O was added until the
solution became cloudy. The solution was cooled in an ice bath and sheet-like crystals
1
formed. Collected a total 10.5801 g (93.4% yield) of 7 from three crops of crystals. H
NMR (acetone-d6) 8.21 (broad singlet, 1H), 7.85 (doublet, 2H), 7.66 (doublet, 2H), 4.12
(doublet, 2H), 3.68 (singlet, 3H).
(4-bromobenzyl-1-carbonyl)-glycine (Bba-Gly-OH) (8). Saponification of
10.5801 g (39 mmol) of 7 was achieved by stirring it in a solution of 100 mL MeOH and
40 mL H2O containing 6.54 g (117 mmol) KOH for 3 hours. The solution was acidified
with HCl and a white solid slowly precipitated. The solid material was filtered and
1
washed with DI H2O. Obtained 8.5323 g (85.0% yield) of 8. H NMR (acetone-d6) 8.12
(broad singlet, 1H), 7.85 (doublet, 2H), 7.66 (doublet, 2H), 4.12 (doublet, 2H).
(4-bromobenzyl-1-carbonyl)-glycyl-tyrosine methyl ester (Bba-Gly-Tyr-
OMe) (9). The coupling procedure for the synthesis of 7 was used on 0.9287 g (3.6
mmol) 8. The product was recrystallized from hexanes/EtOAc to yield 0.9637 g (61.5%
1
yield) of 9. H NMR (acetone-d6) 8.20 (singlet, <1H), 8.08 (broad doublet, 1H), 7.85
(doublet, 2H), 7.67 (doublet, 2H), 7.42 (broad doublet, 1H), 7.00 (doublet, 2H), 6.69
(doublet, 2H), 4.65 (multiplet, 1H), 4.03 (doublet, 2H), 3.63 (singlet, 3H), 2.95
(multiplet, 2H). HRMS (FAB) m/z = 435 (M+ for 79Br) and m/z = 437 (M+2 for 81Br).
(4-bromobenzyl-1-carbonyl)-glycyl-tyrosine (Bba-Gly-Tyr) (2). The
saponification procedure for the synthesis of 1 was used on 0.9637 g (2.2 mmol) of 9.
1
Obtained 0.5985 g (64.2% yield) of 2. H NMR (200 MHz, acetone-d6) 8.10 (multiplet,

<1H), 7.85 (doublet, J = 9 Hz, 2H), 7.67 (doublet, J = 9 Hz, 2H), 7.36 (doublet, J = 7 Hz,
1H), 7.03 (doublet, J = 9 Hz, 2H), 6.68 (doublet, J = 9 Hz, 2H), 4.68 (multiplet, 1H), 4.04
(doublet, J = 6 Hz, 2H), 3.01 (multiplet, 2H); 13C NMR (300 MHz, acetone-d6) 171.5,
168.4, 166. 0, 154.9, 131.6, 130.3, 129.0, 127.9, 126.0, 124.6, 113.8, 52.0, 41.4, 35.0;
HRMS (FAB, MeOH) m/z = 421.0403 (calc. m/z = 421.0399 for C18H17BrN2O5). UV/Vis
(0.1001 M NaOH) λmax = 243.5 nm, ε243.5 = 24,000; λmax = 295.1 nm, ε295.1 = 1,600; ε308.0
=
800.
(4-bromobenzyl-1-carbonyl)-glycyl-glycine methyl ester (Bba-Gly-GlyOMe)
(10). Followed the coupling procedure for 7 on 4.0224 g (16 mmol) of 8. The product
1
was not massed. It used in the next step of the synthesis without purification. H NMR
(acetone-d6) 8.16 (broad doublet, 1H), 7.86 (doublet, 2H), 7.66 (doublet with broad base,
3H), 4.09 (doublet, 2H), 3.96 (doublet, 2H), 3.64 (singlet, 3H).
(4-bromobenzyl-1-carbonyl)-glycyl-glycine (Bba-Gly-Gly-OH) (11). The
saponification procedure for the synthesis of 1 was used on 5.2223 g of 10. Obtained
1
4.5348 g (90.7% combine yield for coupling and deprotection steps) of 11. H NMR
(acetone-d6) 7.88 (doublet, 2H), 7.68 (doublet, 2H), 4.09 (doublet, 2H), 3.97 (doublet,
2H).
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-tyrosine methyl ester (Bba-Gly-
Gly-Tyr-OMe) (12). The coupling procedure for the synthesis of 7 was followed on
4.5348 g (14 mmol) of 11. Recrystallized product from acetone/MeOH to obtain 3.6289
1
g (51.2% yield) of 12. H NMR (CD3OD) 7.79 (doublet, 2H), 7.62 (doublet, 2H), 7.00
(doublet, 2H), 6.68 (doublet, <2H), 4.59 (multiplet, 1H), 4.03 (singlet, 2), 3.87 (doublet,
2H), 3.64 (singlet, 3H), 2.98 (multiplet, 2H).
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-tyrosine (Bba-Gly-Gly-Tyr) (3).
Saponified 12 using procedure for the synthesis of 1 on 3.6289 g (7.4 mmol) of 12.
1
Obtained 1.5209 g (43.1% yield) of 3. H NMR (250 MHz, CD3OD) 7.98 (broad
doublet, J = 8 Hz, <1H), 7.79 (doublet, J = 8 Hz, 2H), 7.62 (doublet, J = 8 Hz, 2H), 7.03

(doublet, J = 8 Hz, 2H), 6.68 (doublet, J = 8 Hz, 2H), 4.59 (multiplet, 1H), 4.07 (doublet,
J = 7 Hz, 2H), 3.91 (doublet, J = 7 Hz, 2H), 3.02 (multiplet, 2H); HRMS (FAB, MeOH)
m/z = 478.0618 (calc. m/z = 478.0614 for C20H20BrN3O6). UV/Vis (0.1001 M NaOH) λmax
= 243.7 nm, ε243.7 = 24,000; λmax = 295.4 nm, ε295.4 = 1,200; ε308.0 = 200.
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycine methyl ester (Bba-Gly-Gly-
Gly-OMe) (14). The coupling method for the synthesis of 7 was followed using 4.1389
g (13 mmol) of 11. Obtained 4.20 g (82.8% yield) of 14. An 1H NMR spectrum was not
taken of this compound.
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycine (Bba-Gly-Gly-Gly-OH)
(15). Followed the de-esterification procedure for the synthesis of 1 using 4.20 g (11
mmol) of 14. The product was not massed and was used in the next step of the reaction
1
without purification. H NMR (DMSO-d6) 8.90 (triplet, 1H), 8.26 and 8.17 (two
overlapping triplets, 2H), 7.83 (doublet, 2H), 7.69 (doublet, 2H), 3.90 (doublet, integral
obscured by H2O), 3.75 (multiplet, integral obscured by H2O).
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycyl-tyrosine methyl ester (Bba-
Gly-Gly-Gly-Tyr-OMe) (16). The procedure for the synthesis of 7 was followed using
all the material from the synthesis of 15, assuming a 100% yield from the previous step
(i.e. 4.0474 g (11 mmol) of 15), and using DMAP as the catalyst. Crystallized product
1
from EtOAc to obtain 3.4665 g (58.0% yield for two steps) of 16. H NMR (CD3OD)
7.79 (doublet, 2H), 7.62 (doublet, 2H), 6.97 (doublet, 2H), 6.68 (doublet, 2H), 4.56
(multiplet, 1H), 4.08 (singlet, 2H), 3.88 (multiplet, 4H), 3.63 (singlet, 3H), 2.93
(multiplet, 2H).
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycyl-tyrosine (Bba-Gly-Gly-Gly-
Tyr) (4). Used the saponification procedure for the synthesis of 8 on 3.4665 g (6.3
1
mmol) of 16. Obtained 2.6415 g (78.2% yield) of 4. H NMR (200 MHz, CD3OD) 7.68
(doublet, J = 8 Hz, 2H), 7.52 (doublet, J = 8 Hz, 2H), 6.90 (doublet, J = 8 Hz, 2H), 6.56
(doublet, J = 8 Hz, 2H), 4.44 (multiplet, 1H), 3.96 (singlet, 2H), 3.77 (multiplet, 4H),

2.85 (multiplet, 2H); HRMS (FAB, MeOH) m/z = 535.0811 (calc. m/z = 535.0828 for
C22H23BrN4O7). UV/Vis (0.1001 M NaOH) λmax = 243.8 nm, ε243.8 = 27,000; λmax = 292.6
nm, ε292.6 = 2,000; ε308.0 = 500.
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycyl-glycine methyl ester (Bba-
Gly-Gly-Gly-Gly-OMe) (17). Followed the coupling procedure described for the
synthesis of 7 using 6.3666 g (17 mmol) of 15, 1.4037 g (11 mmol) DMAP in addition to
1
2.5602 g (19 mmol) HOBt. Obtained 4.6246 g (61% yield) of 17. H NMR (DMSO-d6)
8.92 (broad singlet, 1H), 8.25 (broad multiplet, 3H), 7.82 (doublet, 2H), 7.69 (doublet,
2H), 3.91 (broad singlet, 2H), 3.82 (doublet, 2H), 3.74 (broad singlet, 4H), 3.60 (singlet,
3H).
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycyl-glycine (Bba-Gly-Gly-Gly-
Gly-OH) (18). Saponified 17 using procedure for synthesis of 8 using 4.6246 g (10
mmol) of 17. Obtained 3.6440 g (81.4% yield) of 18. No 1H NMR of this compound
was taken.
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycyl-glycine-tyrosine methyl
ester. (Bba-Gly-Gly-Gly-Gly-Tyr-OMe) (19). The coupling procedure for the synthesis
of 17 was followed using 3.6440 g (8.4 mmol) of 18. Obtained 1.8440 g (39.5% yield) of
19. This compound was not analyzed by 1H NMR and was used in the next step of the
reaction.
(4-bromobenzyl-1-carbonyl)-glycyl-glycyl-glycyl-glycine-tyrosine (Bba-Gly-
Gly-Gly-Gly-Tyr) (5). Followed the procedure for the de-esterification of 8 using
1.8440 g (3.0 mmol) of 19. The reaction did not go to completion, but some product was
1
formed (5) -- the material obtained was nearly insoluble in all solvents. H NMR (hot
DMSO-d6) 9.79 (triplet, .5H), 8.22 and 8.13 (two overlapping triplets, 2.5H), 7.81
(doublet, 2H), 7.69 (doublet, 2H), 6.98 (doublet, .3H), 6.63 (doublet, .3H), 3.90 (doublet,
2H), 3.82 (doublet, 1H), 3.74 (triplet of overlapping resonances, 5H), 3.60 (singlet, 1H).

References
(1) Forbes, M. D. E.; Peterson, J.; Breivogel C. S. Closs, G. L. Rev. Sci. Instrum. 1991,
62, 2662.
(2) Closs, G. L.; Forbes, M. D. E. J. Phys. Chem. 1991, 95, 1924.
(3) Closs, G. L.; Forbes, M. D. E.; Norris, J. R. Jr. J. Phys. Chem. 1987, 91, 3592.