Cysteinoyl- and Cysteine-containing Dipeptidoylbenzotriazoles with Free Sulfhydryl Groups: Easy Access to N-terminal and Internal Cysteine Peptides

Article abstract of DOI:10.1111/j.1747-0285.2011.01303.x

N-Protected cysteines 4a-c each with a free sulfhydryl group were prepared in 70-75% yields by treatment of l-cysteine with 1-(benzyloxycarbonyl) benzotriazole (Cbz-Bt) 1a, N-(tert-butyloxy-carbonyl)benzotriazole (Boc-Bt) 1b, and 1-(9-fluorenylmethoxy-carbonyl)benzotriazole (Fmoc-Bt) 1c, respectively. N-Protected, free sulfhydryl cysteines 4a-c were then converted into the corresponding N-protected, free sulfhydryl cysteinoylbenzotriazoles 7a-c (70-85%), which on treatment with diverse amino acids and dipeptides afforded the corresponding N-protected, free sulfhydryl N-terminal cysteine dipeptides 8a-e and tripeptides 8f-h in 73-80% yields. N-Protected, free sulfhydryl cysteine-containing dipeptides 9a,b were converted into the corresponding N-protected, free sulfhydryl dipeptidoylbenzotriazoles 10a,b (69-81%), which on treatment with amino acids, dipeptides, and a tripeptide afforded internal cysteine tripeptides 11a-c, tetrapeptides 11d,e and pentapeptide 11f, each containing a N-protected, free sulfhydryl groups in 70-90% yields under mild conditions. Treatment of N-protected, free sulfhydryl cysteinoylbenzotriazole 7a with diamines 12a,b afforded directly the cysteine-containing disulfide-bridged cyclic peptides 14a,b in 50% yields. Novel and stable N-protected, free sulfhydryl, cysteine-containing dipeptidoylbenzotriazoles formed internal cysteine containing N-protected free sulfhydryl tripeptides, tetrapeptides, and pentapeptide under mild reaction conditions in good yields that can be useful in the syntheses of other naturally occurring or biologically active cysteine-containing peptides.

Full text of DOI:10.1111/j.1747-0285.2011.01303.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01303.x
Chem Biol Drug Des 2012; 80: 194–202
Research Article
Cysteinoyl- and Cysteine-containing
Dipeptidoylbenzotriazoles with Free Sulfhydryl
Groups: Easy Access to N-terminal and Internal
Cysteine Peptides
Tarek S. Ibrahim1,2, Srinivasa R. Tala1,†
Said A. El-Feky2, Zakaria K. Abdel-Samii2
and Alan R. Katritzky1,3,
,
Cysteine peptides are diagnostically and therapeutically important
in many areas of biomedical research (1–6) and thus attractive tar-
gets for drug discovery. Consequently, interest in cysteine peptide
synthesis has increased significantly (7–9), including the preparation
of unsymmetrical disulfides (10,11), formation of oligodeoxynucleo-
tide (ODN)-peptide covalent linkages (12), and the synthesis of
cyclic peptides (13).
*
1Department of Chemistry, Center for Heterocyclic Compounds,
University of Florida, Gainesville, FL 32611-7200, USA
2Faculty of Pharmacy, Department of Pharmaceutical Organic
Chemistry, Zagazig University, Zagazig-44519, Egypt
3Department of Chemistry, King Abdulaziz University, Jeddah 21589,
Saudi Arabia
N-Terminal cysteine peptides with the cysteine residues in the free
thiol form are frequently needed (14,15). Syntheses of such cysteine
peptides usually require initial SH-protection or regioselective intra-
or intermolecular disulfide bridge formation (14,15), and such maxi-
mum protection has been the norm during the coupling steps in
solid-phase peptide synthesis (16,17).
*Corresponding author: Alan R. Katritzky, katritzky@chem.ufl.edu
Present address: Department of Medicinal Chemistry, College of
Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA.
N-Protected cysteines 4a–c each with a free sulfhy-
dryl group were prepared in 70–75% yields by
treatment of L-cysteine with 1-(benzyloxycarbonyl)
benzotriazole (Cbz-Bt) 1a, N-(tert-butyloxy-car-
bonyl)benzotriazole (Boc-Bt) 1b, and 1-(9-fluorenyl-
N-Cbz, N-Fmoc, and N-Boc-L-cysteine units are valuable building
blocks for solid-phase peptide synthesis (18–20). They are avail-
able (i) in the form of disulfides which may subsequently be
cleaved to the free SH group or (ii) with SH protected with Fmoc
(18–20). Cysteine residues with SH protected by groups such as
Fmoc or Boc are prone to side reactions such as (i) b-elimination
leading to the formation of dehydroalanine derivatives under basic
reaction conditions, followed by subsequent conjugate nucleophilic
addition, for example, of piperidine (1) (ii) oxidation (18,21), (iii)
alkylation (18,21), and (iv) acyl migration from sulfur to nitrogen
(22).
methoxy-carbonyl)benzotriazole
(Fmoc-Bt)
1c,
respectively. N-Protected, free sulfhydryl cysteines
4a–c were then converted into the corresponding
N-protected, free sulfhydryl cysteinoylbenzotriaz-
oles 7a–c (70–85%), which on treatment with
diverse amino acids and dipeptides afforded the
corresponding N-protected, free sulfhydryl N-ter-
minal cysteine dipeptides 8a–e and tripeptides 8f–h
in 73–80% yields. N-Protected, free sulfhydryl cys-
teine-containing dipeptides 9a,b were converted
into the corresponding N-protected, free sulfhydryl
dipeptidoylbenzotriazoles 10a,b (69–81%), which
on treatment with amino acids, dipeptides, and a
tripeptide afforded internal cysteine tripeptides
11a–c, tetrapeptides 11d,e and pentapeptide 11f,
We now report N-protected, SH-unprotected cysteinoylbenzotriaz-
oles and cysteine-containing dipeptidoylbenzotriazoles which allow
the efficient synthesis of N-terminal cysteine-containing peptides
and internal cysteine-containing peptides, respectively, both with
free sulfhydryl groups. Subsequent cyclization under mild conditions
in the case of bis-cysteine-containing peptides forms disulfide-
bridged cyclic peptides. We employ three different N-protecting
groups (Cbz, Boc, and Fmoc) for the synthesis of the cysteine pep-
tide sequences, all of which were compatible with reaction condi-
tions convenient for peptide synthesis.
each containing
groups in 70–90% yields under mild conditions.
Treatment of N-protected, free sulfhydryl
a N-protected, free sulfhydryl
cysteinoylbenzotriazole 7a with diamines 12a,b
afforded directly the cysteine-containing disulfide-
bridged cyclic peptides 14a,b in 50% yields.
Key words: acylation, benzotriazole methodology, cysteine, N-pro-
tected(a-aminoacyl)benzotriazole, small peptides
Materials and Methods
Received 26 July 2011, revised 5 December 2011 and accepted for pub-
lication 5 December 2011
Melting points were determined on a capillary point apparatus
equipped with a digital thermometer and are uncorrected. NMR
194

Cysteinoyl- and Cysteine-containing Dipeptidoylbenzotriazoles with Free Sulfhydryl Groups
spectra were recorded in CDCl3 or DMSO-d6 with TMS for 1H
N-Cbz-L-Cys-OH (4a) (27)
1
(300 MHz) and 13C (75 MHz) as an internal reference. Elemental
analyses were performed on a Carlo Erba–1106 instrument (Carlo
Erba Reagents, Val de Reuil, France). CH2Cl2 was dried and distilled
over CaH2, whereas THF was used after distillation over Na-benzo-
phenone. Mass spectrometry was carried out on Agilent 6210 TOF-
MS with electrospray ionization (ESI).
White microcrystals, yield 75%, mp 67–68 ꢀC; H NMR (DMSO-d6)
d 12.96 (s, 1H), 8.03 (s, 1H), 7.35 (s, 5H), 5.04 (s, 2H), 4.27 (s, 1H),
3.14 (d, J = 13.5 Hz, 1H), 2.91 (t, J = 15 Hz, 1H), 1.23 (s, 1H); 13C
NMR (DMSO-d6) d 172.1, 156.3, 137.0, 128.5, 128.2, 128.0, 65.8,
56.7, 25.7. Anal. Calcd for C11H13N4O4S: C, 51.75; H, 5.13; N, 5.49.
Found: C, 51.65; H, 4.90; N, 5.36.
Preparation of N-Cbz, N-Boc-, and N-Fmoc-1H-
benzotriazoles 1a–c
N-Cbz, N-Boc-, and N-Fmoc-1H-Benzotriazoles 1a–c were prepared
according to literature procedures (23,26).
N-Boc-L-Cys-OH (4b)
Oil, yield 71%, (Lit., (28) 75)78 ꢀC); H NMR (DMSO-d6) d 12.81 (br
s, 1H), 7.15 (d, J = 8.1 Hz, 1H), 4.18 (s, 1H), 3.08 (d, J = 12.6 Hz,
1H), 2.88 (t, J = 11.1 Hz, 1H), 1.38 (s, 9H); 13C NMR (DMSO-d6) d
172.5, 155.4, 78.4, 52.8, 28.2. Anal. Calcd for C8H15NO4S: C, 43.42;
H, 6.83; N, 6.33. Found: C, 43.77; H, 6.54; N, 6.10.
1
N-Cbz-1H-Benzotriazole (1a) (23,24)
1a was synthesized according to literature method. (23,24) White
microcrystals, yield 85%, mp 108–110 ꢀC (lit., (23) 108.0–110.0 ꢀC);
1H NMR (CDCl3) d 8.12 (t, J = 9.3 Hz, 2H), 7.68–7.60 (m, 1H), 7.60–
7.54 (m, 2H), 7.54–7.39 (m, 4H), 5.64 (s, 2H); 13C NMR (DMSO–d6)
d 148.2, 145.1, 134.4, 131.2, 130.5, 128.8, 128.7, 128.6, 125.9,
120.1, 113.3, 69.9.
N-Fmoc-L-Cys-OH (4c)
White microcrystals, yield 70%, mp 134–136 ꢀC (lit., (29) 140–
1
142 ꢀC); H NMR (DMSO-d6) d 12.80 (br s, 1H), 7.90 (d, J = 7.2 Hz,
2H), 7.90 (d, J = 7.2 Hz, 2H), 7.90 (d, J = 7.2 Hz, 2H), 7.79–7.60 (m,
3H), 7.43 (t, J = 7.2 Hz, 2H), 4.32 (d, J = 6 Hz, 2H), 4.26 (d,
J = 6.3 Hz, 1H), 4.20–4.08 (m, 1H), 2.90 (d, J = 8.1 Hz, 1H), 2.84–
2.65 (m, 1H), 1.26 (br s, 1H); 13C NMR (DMSO-d6) d 171.4, 169.1,
156.5, 143.8, 140.7, 127.6, 127.1, 125.2, 120.1, 65.8, 54.2, 46.6,
25.7.
N-Boc-1H-Benzotriazole (1b) (25,26)
1b was synthesized according to literature method (25,26). White mi-
crocrystals, yield 50%, mp 70.0–72.0 ꢀC (lit., (25,26) 62.0–63.0 ꢀC);
1H NMR (CDCl3) d 8.22–8.02 (m, 2H), 7.64 (t, J = 7.8 Hz, 1H), 7.48 (t,
J = 7.8 Hz, 1H), 1.77 (s, 9H); 13C NMR (CDCl3) d 146.8, 146.1, 131.8,
130.0, 125.6, 120.4, 113.7, 87.1, 28.2.
S-Acylation of N-Cbz-, N-Boc-, and N-Fmoc-L-
cysteines 6a–c
To a solution of N-Cbz-, N-Boc-, and N-Fmoc-L-cysteines 4a–c.
(1 mmol) in THF (5 mL) was added a solution of N-Cbz, N-Boc-,
and N-Fmoc-L-cysteines 4a–c (1 mmol) in THF (5 mL) in the pres-
ence of KHCO3 (1 mmol). The heterogeneous mixture was then
stirred at room temperature for 12 h. THF was evaporated, and
the solution was acidified with HCl, and the solid formed was
taken in ethyl acetate and washed with HCl (1N, 3·) and brine
(3·) and then dried over sodium sulfate. Evaporation of ethyl ace-
tate under reduced pressure afforded N-protected S-acyl cysteines
6a–c.
N-Fmoc-1H-Benzotriazole (1c)
A
mixture of (9H-fluoren-9-yl)methyl chloroformate (1 g,
3.87 mmol) and benzotriazole (0.92 g, 7.74 mmol) in CH2Cl2
(20 mL) was stirred for 3 h at 10 ꢀC. The precipitate formed was
filtered off and discarded. The filtrate was evaporated under
reduced pressure to give a crude product, which was washed
with diethyl ether and recrystallized from CH2Cl2-hexanes to
afford N-Fmoc-1H-benzotriazole. White microcrystals, yield 88%,
mp 90–91 ꢀC; 1H NMR (DMSO-d6) d 8.09–8.18 (m, 1H), 7.91 (d,
J = 7.2 Hz, 2 H), 7.81 (d, J = 7.2, 2H), 7.51–7.39 (4H, m), 7.38–
7.29 (2H, m), 7.23 (br s, 1H), 5.10 (d, J = 5.1 Hz, 2H), 4.58 (br s,
1H); 13C NMR (DMSO-d6) d 148.0, 145.0, 143.1, 140.9, 130.8,
130.1, 127.9, 127.3, 125.8, 125.0, 120.2, 119.9, 113.0, 69.5, 46.2.
Anal. Calcd for C21H15N3O2: C, 73.89; H, 4.43; N, 12.31. Found:
C, 73.84; H, 4.06; N, 12.29.
N-Cbz-L-Cys(S-4-NO2-Bz)-OH (6a)
White microcrystals, yield 80%, mp 155–156 ꢀC; H NMR (DMSO-
1
d6) d 7.60 (d, J = 6.9 Hz, 1H), 7.37 (s, 9H), 5.06 (s, 2H), 4.14 (d,
J = 4.2 Hz, 1H), 2.87 (br s, 1H), 2.74 (br s, 1H); 13C NMR (DMSO-d6)
d 197.6, 172.3, 155.6, 137.7, 137.0, 129.3, 128.4, 128.0, 127.8,
126.3, 125.2, 65.5, 53.9, 30.7. Anal. Calcd for C8H15NO4S.H2O: C,
51.18; H, 4.29; N, 6.63. Found: C, 51.26; H, 4.67; N, 6.60.
Synthesis of N-Cbz-, N-Boc-, and N-Fmoc-L-
cysteines 4a–c
To a solution of L-cysteine 2 (1 mmol) and triethylamine (1 mmol) in
CH3CN-H2O (3:1, 8 mL) was added the corresponding N-Cbz, N-Boc-,
and N-Fmoc-1H-benzotriazoles 1a–c (1 mmol). The mixture was stir-
red at room temperature for 2 h. Solvent was removed under
reduced pressure, and ethyl acetate (10 mL) was added. The
organic layer was washed with 2 N HCl and brine. Evaporation to
the solvent followed by recrystalization (AcOEt: hexanes = 3:1) gave
N-Cbz-, N-Boc-, and N-Fmoc-L-cysteine 4a–c.
N-Boc-L-Cys(S-4-NO2-Bz)-OH (6b)
White microcrystals, yield 85%, mp 175–177 ꢀC; H NMR (DMSO-
d6) d 7.69 (s, 2H), 7.45 (s, 3H), 4.20 (br s, 1H), 3.65 (br s, 1H), 3.28
(br s, 1H), 1.22–1.58 (m, 9H); 13C NMR (DMSO-d6) d 199.8, 171.5,
155.7, 155.5, 137.1, 129.1, 128.3, 127.8, 126.5, 78.1, 65.4, 29.6,
28.2. Anal. Calcd for C15H18N2O7S: C, 46.64; H, 4.90; N, 7.56.
Found: C, 46.26; H, 4.51; N, 7.25.
1
Chem Biol Drug Des 2012; 80: 194–202
195

Ibrahim et al.
N-Fmoc-L-Cys(S-4-OMe-Bz)-OH (6c)
N-(N¢-Boc-L-Cys)-1H-benzotriazole (7b)
White microcrystals, yield 80%, mp 173–175 ꢀC; H NMR (DMSO-
d6) d 7.91 (s, 2H), 7.44 (s, 2H), 7.23 (d, J = 3.5 Hz, 1H), 4.19 (s,
1H,), 3.00–2.60 (m, 2H), 1.77 (d, J = 12.0 Hz, 1H), 1.37 (s, 9H); 13C
NMR (DMSO-d6) d 170.7, 156.6, 155.5, 131.2, 128.6, 126.8, 120.2,
113.9, 79.0, 52.9, 28.1, 27.6. Anal. Calcd for C14H18N4O3S: C, 52.16;
H, 5.63; N, 17.38. Found: C, 52.34; H, 5.75; N, 17.50.
1
1
White microcrystals, yield 78%, mp 182–184 ꢀC; H NMR (DMSO-
d6) d 8.30 (dd, J = 8.4, 5.4 Hz, 1H), 8.18 (d, J = 9.3 Hz, 1H), 7.86–
7.94 (m, 2H), 7.86–7.78 (m, 1H), 7.76–7.62 (m, 2H), 7.49–7.38 (m,
3H), 7.34 (t, J = 6.3 Hz, 2H), 7.21 (d, J = 8.7 Hz, 1H), 4.50–4.10 (m,
3H), 3.92 (s, 3H), 3.37 (br s, 1H), 3.28–3.09 (m, 1H), 3.08–2.85 (m,
1H); 13C NMR (DMSO-d6) d 211.8, 181.2, 167.1, 154.6, 151.7, 147.4,
138.9, 138.3, 138.1, 137.6, 135.8, 130.4, 67.6, 62.1, 57.6, 54.3, 27.5.
Anal. Calcd for C26H23NO6S: C, 65.39; H, 4.85; N, 2.93. Found: C,
65.37; H, 4.58; N, 3.26.
General procedure for N-terminal cysteine-
containing N-Cbz-, N-Boc-, and N-Fmoc-
dipeptides 8a–e, (8a + 8a¢) and tripeptides 8f–h
N-(N¢-Cbz-, N¢-Boc- and N¢-Fmoc-L-cys)-1H-benzotriazoles 7a–c (1
equiv.) were added at room temperature to a solution of amino acid
or dipeptide (1 equiv.) in MeCN ⁄ H2O (7:3 mL) in the presence of tri-
ethylamine (1 equiv.). The reaction mixture was stirred at room tem-
perature until the starting material was completely consumed, as
observed by TLC using hexanes-ethyl acetate (2:1) as eluent. Aq.
6 N HCl solution was then added, and the solvent was removed
under reduced pressure. The residue was dissolved in ethyl acetate
(20 mL), and the organic extract was washed with 6 N HCl solution,
sat. NaCl solution and dried over anhydrous MgSO4. Evaporation of
the solvent gave the desired products, which were recrystallized
from chloroform- hexanes or ethyl acetate-hexanes.
Synthesis of N-(N¢- Cbz-and N¢-Fmoc-L-Cys)-1H-
benzotriazoles 7a,c
Thionyl chloride (1 mmol) was added to a solution of 1H-benzotriaz-
ole (4 mmol) in anhydrous THF (30 mL) at 25 ꢀC, and the reaction
mixture was stirred for 20 min. N-Cbz- and N-Fmoc-L-cysteines 4a–
c (1 mmol) were added to the reaction mixture, which was stirred
for 2 h at 0–5ꢀC. The white precipitate which formed was filtered
off, and the filtrate was concentrated under reduced pressure. The
residue was diluted with EtOAc (15 mL), and the solution was
washed with sat. Na2CO3 solution (3 · 5 mL), sat. NaCl solution
(5 mL), and dried with (MgSO4). Removal of the solvent under
reduced pressure gave 7a–c, which were recrystallized from
CH2Cl2-hexanes.
N-Cbz-L-Cys-L-Phe-OH (8a) (27)
White microcrystals, yield 80%, mp 131–132ꢀC; H NMR (DMSO-d6)
1
N-(N¢-Cbz-L-Cys)-1H-benzotriazole (7a)
White microcrystals, yield 70%, mp 155–156 ꢀC (lit., (30) 144–
147 ꢀC); 1H NMR (DMSO-d6) d 8.41 (d, J = 7.2 Hz, 1H), 8.22 (d,
J = 8.1 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 7.82 (t, J = 8.1 Hz, 1H),
7.74–7.60 (m, 1H), 7.54–7.30 (m, 5H), 7.02 (br s, 1H), 6.05–5.82 (s,
1H), 4.95–5.20 (s, 2H), 3.61–3.45 (m, 1H), 3.25–3.10 (m, 1H); 13C
NMR (DMSO-d6) d 170.3, 156.1, 145.2, 136.5, 131.2, 130.5, 128.3,
127.9, 127.8, 126.8, 120.1, 113.9, 66.0, 53.2, 37.2. Anal. Calcd for
C17H16N4O3S.1 ⁄ 2H2O: C, 63.56; H, 4.67; N, 12.35. Found: C, 63.78;
H, 4.67; N, 12.65.
d 12.88 (s, 1H), 8.24–8.18 (m, 1H), 7.75–7.52 (m, 1H), 7.30–7.18 (m,
6H), 7.30–7.10 (m, 4H), 5.05 (s, 2H), 4.55–4.40 (m, 1H), 4.32 (br s,
1H), 3.24–2.90 (m, 3H), 2.88–2.72 (m, 1H); 13C NMR (DMSO-d6) d
172.5, 170.0, 155.9, 137.2, 136.8, 129.2, 128.3, 128.1, 127.8, 127.7,
126.4, 65.6, 53.6, 53.5, 36.5, 22.7. Anal. Calcd for C20H22N2O5S: C,
59.69; H, 5.51; N, 6.96. Found: C, 59.47; H, 5.57; N, 7.01.
N-Cbz-L-Cys-DL-Phe-OH (8a + 8a¢) (27)
1
White microcrystals, yield 80%; H NMR (DMSO-d6) d 12.90 (br s,
1H), 7.42–7.16 (m, 12H), 5.10–5.00 (m, 2H), 4.55–4.42 (m, 1H),
4.40–4.30 (m, 1H), 3.20–2.73 (m, 4H), 1.23 (s, 1H); 13C NMR
(DMSO-d6) d 172.6, 170.4, 170.1, 156.7, 156.0, 138.4, 137.3, 136.9,
129.2, 128.9, 128.4, 128.2, 127.8, 127.7, 126.8, 126.5, 69.7, 69.1,
65.7, 65.0, 54.4, 53.6, 36.6, 35.4, 33.6. Anal. Calcd for C20H22N2O5S:
C, 59.69; H, 5.51; N, 6.96. Found: C, 59.75; H, 5.60; N, 7.00.
N-(N¢-Fmoc-L-Cys)-1H-benzotriazole (7c)
White microcrystals, yield 85%, mp 169–171ꢀC; H NMR (DMSO-d6)
1
d 8.08–7.80 (m, 5H), 7.72 (t, J = 6.6 Hz, 2H), 7.58–7.24 (m, 6H),
4.59–4.22 (m, 3H), 3.68 (d, J = 3.3 Hz, 1H), 3.10–3.24 (m, 1H),
3.06–2.88 (m, 1H); 13C NMR (DMSO-d6) d 172.3, 171.5, 156.1,
143.8, 140.8, 128.0, 127.7, 127.1, 125.3, 120.1, 65.8, 53.1, 46.7,
31.4. Anal. Calcd for C14H18N4O3S: C, 64.90; H, 5.11, N, 12.60.
Found: C, 64.85; H, 4.99; N, 12.97.
N-Fmoc-L-Cys-L-Phe-OH (8b)
1
White microcrystals, yield 75%, mp 145–146 ꢀC; H NMR (DMSO-
d6) d 13.00 (br s, 1H), 8.39 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 7.8 Hz,
2H), 7.71 (d, J = 8.7 Hz, 1H), 7.65 (dd, J = 6.9, 4.2 Hz, 2H), 7.44–
7.15 (m, 9H), 4.60–4.48 (m, 1H), 4.47–4.35 (m, 1H), 4.25–4.10 (m,
2H), 4.03 (dd, J = 14.0, 6.9 Hz, 1H), 3.09 (dd, J = 13.7, 3.3 Hz, 1H),
3.00–2.77 (m, 3H), 2.44 (t, J = 8.4 Hz, 1H,); 13C NMR (DMSO-d6) d
171.8, 171.5, 155.8, 143.8, 140.7, 138.1, 129.3, 128.1, 127.6, 127.1,
126.3, 125.3, 120.1, 65.7, 56.0, 54.4, 46.6, 37.5, 25.7. Anal. Calcd
for C27H26N2O5S: C, 66.10; H, 5.34; N, 5.71. Found: C, 65.97; H,
5.51; N, 5.38.
Synthesis of N-(N¢-Boc-L-Cys)-1H-benzotriazole
(7b)
To Boc-L-Cysteine (0.221 g, 1 mmol), dissolved in dichloromethane
(30 mL), benzotriazole (0.119 g, 1 mmol) and dicyclohexylcarbodii-
mide (0.206 g, 1 mmol) were added. The reaction mixture was stir-
red at 0–5 ꢀC, overnight. Dicyclohexylurea was filtered off, and the
dichloromethane was evaporated. The residue was recrystallized
from dichloromethane (20 mL) and hexanes (25 mL).
196
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Cysteinoyl- and Cysteine-containing Dipeptidoylbenzotriazoles with Free Sulfhydryl Groups
N-Cbz-L-Cys-L-Ser-OH (8c)
(dd, J = 11.4, 9.3 Hz, 1H), 7.40–7.28 (m, 5H), 5.04 (s, 3H), 4.40–
4.14 (m, 2H), 3.73 (d, J = 5.7 Hz, 1H), 3.22–3.08 (m, 1H), 2.96–
2.80 (m, 1H), 1.26 (d, J = 7.2 Hz, 3H), 0.85 (d, J = 7.5 Hz, 1H);
13C NMR (DMSO-d6) d 173.9, 172.2, 168.2, 156.1, 136.9, 128.3,
127.8, 127.7, 65.6, 53.9, 53.1, 47.5, 41.9, 17.3. Calcd for
C16H21N3O6S: C, 50.12; H, 5.52; N, 10.96. Found: C, 49.81; H,
5.65; N,11.01.
Oil. Yield 73%; 1H NMR (DMSO-d6) d 8.24 (s, 1H), 7.50–7.20 (m,
6H), 5.04 (s, 2H), 4.50 (s, 1H), 4.13 (s, 1H), 3.62 (br s, 1H), 3.59–
3.40 (m, 1H), 3.18 (br s, 1H), 3.08 (d, J = 6.9 Hz, 1H), 2.97 (br s,
1H), 1.18 (t, J = 7.2 Hz, 1H); 13C NMR (DMSO-d6) d 181.0, 180.8,
166.8, 147.7, 138.9, 138.3, 138.2, 77.7, 76.6, 70.3, 64.5, 29.0. Anal.
Calcd for C14H18N2O6S: C, 49.11; H, 5.30; N, 8.18. Found: C, 49.52;
H, 4.89; N, 7.77.
Synthesis of N-(N¢-Cbz- and N¢-Fmoc-a-
dipeptidoyl)-1H-benzotriazoles 10a,b
N-Fmoc-L-Cys-L-Trp-OH (8d)
White microcrystals, yield 77%, mp 136–137 ꢀC; H NMR (DMSO-
1
Thionyl chloride (1 mmol) was added to a solution of 1H-benzotriaz-
ole (4 mmol) in anhydrous THF (30 mL) at 20 ꢀC, and the reaction
mixture was stirred for 20 min. N-Cbz- and N-Fmoc-L-cysteine di-
peptides 9a,b (1 mmol) were added to the reaction mixture at
)30ꢀC, which was stirred for 4 h at )30 ꢀC. The white precipitate
which formed was filtered off, and the filtrate was concentrated
under reduced pressure. The residue was diluted with EtOAc
(15 mL), and the solution was washed with sat. Na2CO3 solution
(3 · 5 mL), sat. NaCl solution (5 mL), and dried with (MgSO4).
Removal of the solvent under reduced pressure gave 10a,b which
were recrystallized from CH2Cl2-hexanes.
d6) d 12.88 (br s, 1H), 10.85 (s, 1H), 8.35 (d, J = 7.8 Hz, 1H),
7.90–7.80 (m, 2H), 7.74–7.50 (m, 4H), 7.50–7.14 (m, 6H), 7.08 (t,
J = 7.2 Hz, 1H), 6.99 (t, J = 6.9 Hz, 1H), 4.49 (dd, J = 7.2, 2.1 Hz,
1H), 4.44–4.32 (m, 1H), 4.22–4.02 (m, 3H), 3.16 (dd, J = 14.7,
3.6 Hz, 1H,), 3.06–2.75 (m, 3H), 2.42 (t, J = 8.1 Hz, 1H); 13C NMR
(DMSO-d6) d 172.1, 171.4, 155.8, 143.7, 140.6, 136.1, 127.6,
127.3, 127.0, 125.3, 123.9, 120.8, 120.0, 118.5, 118.2, 111.3,
110.1, 65.7, 55.4, 54.4, 46.6, 27.8, 25.5. Anal. Calcd for
C29H27N3O5S: C, 65.77; H, 5.14; N, 7.93. Found: C, 65.37; H, 5.58;
N, 7.46.
N-Boc-L-Cys-L-Ala-OH (8e) (31)
Yellow microcrystals, yield 83%, mp 144–145 ꢀC; H NMR (DMSO-
N-(N¢-Cbz-L-Phe-L-Cys)-1H-benzotriazole (10a)
White microcrystals, yield 81%, mp 154–156 ꢀC; H NMR (CDCl3) d
1
1
d6) d 12.62 (s, 1H), 8.40–8.00 (m, 1H), 7.07 (d, J = 8.7 Hz, 1H),
4.42–4.10 (m, 2H), 3.24–3.00 (m, 1H), 2.99–2.64 (m, 1H), 1.80–0.80
(m, 13H); 13C NMR (DMSO-d6) d 174.1, 170.4, 155.6, 78.8, 53.6,
47.9, 28.4, 24.7, 17.4. Anal. Calcd for C11H20N2O5S: C, 45.19; H,
6.90; N, 9.58. Found: C, 45.32; H, 6.48; N, 10.00.
8.19 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.65 (t,
J = 7.2 Hz, 1H), 7.56)7.48 (m, 1H), 7.36)7.22 (m, 12H), 5.33 (d,
J = 8.1 Hz, 1H), 5.09 (s, 2H), 4.54 (t, J = 7.2 Hz, 1H), 3.66)3.12 (m,
4H); 13C NMR (CDCl3) d 172.6, 167.9, 155.7, 146.0, 136.3, 135.1,
131.0, 129.5, 128.8, 128.7, 128.3, 128.2, 127.6, 126.8, 120.6, 114.4,
67.2, 49.6, 37.7, 18.8. Anal. Calcd for C26H25N5O4S: C, 62.01; H,
5.00; N, 13.91. Found: C, 62.32; H, 5.22; N, 14.18.
N-Cbz-L-Cys-Gly-Gly-OH (8f)
White microcrystals, yield 75%, mp 159–161 ꢀC; H NMR (DMSO-
1
d6) d 8.83 (d, J = 8.7 Hz, 1H), 8.68 (d, J = 7.5 Hz, 1H), 8.13 (br s,
1H), 7.42–7.28 (m, 5H), 5.10–4.95 (m, 2H), 4.66–4.53 (m, 1H),
3.64–3.52 (m, 4H), 3.15–3.05 (m, 1H), 2.98–2.82 (m, 1H); 13C NMR
(DMSO-d6) d 172.8, 172.3, 171.1, 155.6, 142.9, 129.3, 128.3,
128.0, 127.8, 126.2, 65.5, 56.2, 41.8, 24.7. Calcd for
C15H19N3O6S.H2O: C, 46.50; H, 5.46; N, 10.85. Found: C, 46.85; H,
5.60; N, 11.12.
N-(N¢-Fmoc-Gly-L-Cys)-1H-benzotriazole (10b)
White microcrystals, yield 69%, mp 140)142 ꢀC; H NMR (DMSO-
1
d6) d 8.41)8.14 (m, 1H), 7.96)7.81 (m, 3H), 7.78)7.58 (m, 4H),
7.50)7.28 (m, 6H), 4.28 (br s, 3H), 3.74 (dd, J = 17.4, 5.1 Hz, 2H),
3.42)3.25 (m, 2H), 1.23 (s, 1H); 13C NMR (DMSO-d6) d 171.8,
169.8, 156.4, 145.7, 143.8, 140.7, 131.2, 130.5, 127.6, 127.4,
1125.2, 124.9, 120.1, 113.9, 65.8, 53.9, 46.6, 46.0, 31.0. Anal. Calcd
for C26H23N5O4S; C, 62.26; H, 4.62; N, 13.96. Found: C, 62.56; H,
4.67; N, 14.34.
N-Cbz-L-Cys-L-Leu-Gly-OH (8g)
White microcrystals, yield 80%, mp 162–164 ꢀC; H NMR (DMSO-
1
d6) d 8.74 (s, 1H), 8.01(d, J = 7.5 Hz, 1H), 7.76 (s, 1H), 7.37 (s, 5H),
5.18–4.90 (m, 2H), 4.25–4.10 (m, 2H), 3.98 (br s, 1H), 3.63–3.46 (m,
2H), 3.12–2.92 (m, 1H), 1.61 (s, 1H), 1.48 (s, 2H), 0.85 (br s,); 13C
NMR (DMSO-d6) d 174.2, 172.9, 170.9, 156.1, 140.7, 127.6, 127.1,
125.3, 65.7, 58.3, 53.3, 46.7, 40.6, 24.9, 24.2, 23.1, 21.5. Anal.
Calcd for C19H27N3O6S: C, 53.63; H, 6.40; N, 9.88. Found: C, 53.86;
H, 6.22; N, 9.49.
Synthesis of internal cysteine containing free
sulfhydryl N-protected tripeptides 11a–c,
tetrapeptides 11d,e, and pentapeptide 11f
N-(N¢-Cbz- and N¢-Fmoc-a-dipeptidoyl)-1H-benzotriazoles 10a,b (1
equiv.) were added at room temperature to a solution of amino acid
or dipeptide or tripeptide (1 equiv.) in MeCN: H2O (7:3 mL) in the
presence of triethylamine (1 equiv.). The reaction mixture was stir-
red at 0–5ꢀC for 2 h. Aq. 6 N HCl solution was then added, and
the solvent was removed under reduced pressure. The residue was
dissolved in ethyl acetate, and the organic layer was washed with
6 N HCl solution, sat. NaCl solution, and dried over anhydrous
MgSO4. Evaporation of the solvent gave the desired products, which
N-Cbz-L-Cys-Gly-L-Ala-OH (8h)
White microcrystals, yield 80%, mp 143–145 ꢀC; H NMR (DMSO-
1
d6) d 12.80 (s, 1H), 8.34 (br s, 1H), 8.06 (d, J = 7.2 Hz, 1H), 7.72
Chem Biol Drug Des 2012; 80: 194–202
197

Ibrahim et al.
were recrystallized from chloroform-hexanes or ethyl acetate-hex-
anes.
65.5, 54.4, 53.9, 50.2, 41.9, 40.3, 37.4, 25.7, 18.1. Anal. Calcd for
C25H30N4O7S: C, 56.59; H, 5.70; N, 10.56. Found: C, 56.91; H, 5.75;
N, 10.83.
N-Cbz-L-Phe-L-Cys-L-Phe-OH (11a)
White microcrystals, yield 90%, mp 179–180 ꢀC; 1H NMR
(DMSO-d6) d 8.51 (d, J = 7.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H),
7.40)7.14 (m, 1H), 6.98 (m, 16H), 5.05)4.82 (m, 3H), 4.68)4.50
(m, 1H), 4.44)4.20 (m, 1H), 3.32)2.90 (m, 4H), 2.75 (dd,
J = 13.8, 10.8 Hz, 2H), 1.25 (br s, 1H); 13C NMR (DMSO-d6) d
171.7, 167.1, 156.6, 138.1, 137.5, 129.8, 129.0, 128.9, 128.4,
128.0, 127.1, 66.1, 54.0, 53.3, 38.2, 37.2, 26.7. Anal. Calcd for
C29H31N3O6S: C, 63.37; H, 5.68; N, 7.64. Found: C, 63.77; H,
5.81; N, 8.01.
N-Cbz-L-Phe-L-Cys-Gly-L-Ala-L-Leu-OH (11f)
White microcrystals, yield 74%, mp 188–189 ꢀC; H NMR (DMSO-
1
d6) d 8.23 (t, J = 5.4 Hz, 1H), 7.96 (d, J = 7.8 Hz, 2H), 7.42 (s,
1H), 7.35 (br s, 5H), 7.23 (br s, 5H), 5.00 (s, 2H), 4.52 (br s, 1H),
4.22 (t, J = 6.3 Hz, 1H), 4.01 (t, J = 7.2 Hz, 1H), 3.78 (d,
J = 5.7 Hz, 2H), 3.18)2.96 (m, 2H), 2.95)2.75 (m, 2H), 1.78 (br s,
1H), 1.45)1.25 (m, 1H), 1.22)1.02 (m, 5H), 0.86 (d, J = 6.9 Hz,
6H); 13C NMR (DMSO-d6) d 172.8, 172.3, 171.1, 168.6, 155.6,
142.9, 137.7, 136.9, 129.3, 128.3, 128.0, 127.8, 126.2, 65.5, 56.2,
53.8, 50.2, 41.8, 37.5, 36.5, 24.7, 18.1, 15.6, 11.3. Anal. Calcd for
C31H41N5O8S: C, 57.84; H, 6.42; N, 10.88. Found: C, 57.98; H,
6.44; N, 11.15.
N-Cbz-L-Phe-L-Cys-L-Trp-OH (11b)
1
White microcrystals, yield 79%, mp 196–198 ꢀC; H NMR (DMSO-
d6) d 10.86 (s, 1H), 8.34 (s, 1H), 8.15 (d, J = 7.2 Hz, 1H), 8.03 (s,
1H), 7.44)7.24 (m, 14H), 6.98 (t, J = 7.2 Hz, 1H), 4.93 (s, 2H),
4.58)4.42 (m, 1H), 4.30 (br s, 1H), 3.24)3.00 (m, 4H), 2.75 (t,
J = 12.3 Hz, 1H); 13C NMR (DMSO-d6) d 173.1, 171.9, 168.8, 168.5,
155.9, 138.1, 136.9, 136.0, 129.2, 128.2, 128.0, 127.6, 127.4, 127.2,
126.2, 123.7, 120.9, 118.3, 118.1, 111.3, 109.6, 65.2, 56.1, 53.0,
42.1, 41.7, 37.4, 27.1. Anal. Calcd for C31H32N4O6S: C, 63.25; H,
5.48; N, 9.22. Found: C, 63.55; H, 5.78; N, 9.52.
General procedure for disulfide-bridged
cysteine peptides 14a,b
N-(N¢-Cbz-L-Cys)-1H-benzotriazole 7a (0.712 g, 2 mmol) was added
at room temperature to a solution of [1,4-butanediamine (0.088 g,
1 mmol) or 1,6-hexanediamine (0.116 g, 1 mmol)] in a solution of
THF in the presence of triethylamine (0.2 mL). The reaction mixture
was then stirred at room temperature until the starting material
was completely consumed, as observed by TLC using hexanes-ethyl
acetate (2:1) as development solvent. THF was removed under
reduced pressure, and the residue was washed with 2 N HCl solu-
tion, diethyl ether (three times), and recrystallized from methanol-
diethyl ether.
N-Fmoc-Gly-L-Cys-L-Leu-OH (11c)
White microcrystals, yield 70%, mp 162–164 ꢀC. H NMR (DMSO-
1
d6) d 8.74 (d, J = 7.2 Hz, 1H), 7.95 (d, J = 6.0 Hz, 1H), 7.84 (d,
J = 7.5 Hz, 2H), 7.74 (dd, J = 7.2, 3.6 Hz, 2H), 7.37 (t, J = 7.2 Hz,
2H), 7.29 (t, J = 7.2 Hz, 2H), 4.35)4.20 (m, 1H), 4.11 (s, 2H),
3.65)3.40 (m, 2H), 3.15)2.85 (m, 4H), 1.69)1.55 (m, 1H), 1.55)1.38
(m, 2H), 7.37 (t, J = 6.9 Hz, 6H); 13C NMR (DMSO-d6) d 172.4,
171.2, 167.5, 146.1, 140.4, 127.3, 126.9, 126.8, 125.0, 124.9, 119.9,
53.5, 51.8, 46.5, 41.0, 36.0, 34.7, 24.2, 23.1, 21.4. Anal. Calcd for
C26H31N3O6S: C, 60.80; H, 6.08; N, 8.18. Found: C, 61.21; H, 6.12;
N, 8.43.
Dibenzyl((4R,13R)-5,12-dioxo-1,2-dithio-6,11-
diazacyclopentadecane-4,13-diyl)dicarbamate
monohydrate (14a)
1
White microcrystals, yield 50%, mp 156–158 ꢀC; H NMR (DMSO-
d6) d 8.60–7.80 (m, 4H), 7.60–7.25 (m, 10H), 5.03 (s, 4H), 4.50–
4.22 (m, 1H), 4.15–3.95 (m, 1H), 3.60–3.98 (m, 8H), 1.82 (s, 1H),
1.60–1.39 (m, 5H); 13C NMR (DMSO-d6) d 173.1, 173.0, 170.2,
155.9, 155.5, 155.4, 139.5, 137.2, 128.3, 127.7, 124.6, 115.1, 65.4,
55.5, 53.1, 43.1, 38.2, 25.2, 24.6. HRMS (ESI). Calcd for
C26H32N4O6S: [M+Na]+ 583.689. Found: 583.200. Anal. Calcd for
C26H32N4O6.H2O:C, 52.33; H, 6.08; N, 9.39. Found: C, 52.44; H,
6.34; N, 9.52.
N-Cbz-L-Phe-L-Cys-Gly-Gly-OH (11d)
White microcrystals, yield 84%, mp 186–187 ꢀC; 1H NMR (CDCl3)
7.65 (s, 1H), 7.64–7.24 (m, 13H), 5.05)4.90 (m, 2H), 5.90)4.80 (m,
1H), 4.50 (br s, 1H), 4.04)3.72 (m, 6H); 13C NMR (CDCl3) d 173.2,
170.9, 156.9, 136.7, 136.2, 129.4, 128.6, 128.2, 127.9, 127.0, 67.2,
56.6, 42.9, 41.5, 38.2, 29.9. Anal. Calcd for C24H28N4O7S: C, 55.80;
H, 5.46; N, 10.85. Found: C, 56.11; H, 5.48; N, 11.21.
Dibenzyl((4R,15R)-5,14-dioxo-1,2-dithio-6,13-
diazacyclopentadecane-4,15-diyl)dicarbamate
(14b)
1
N-Cbz-L-Phe-L-Cys-Gly-L-Ala-OH (11e)
White microcrystals, yield 50%, mp 167–169 ꢀC; H NMR (DMSO-
1
White microcrystals, yield 80%, mp 172–174 ꢀC; H NMR (DMSO-
d6) d 8.05 (br s, 4H), 7.96–7.84 (m, 2H), 7.56–7.41 (m, 3H), 7.36 (br
s, 5H), 5.95–5.52 (m, 4H), 5.12–4.98 (m, 2H), 4.26 (t, J = 7.2 Hz,
1H), 3.12–3.00 (m, 3H), 2.92–2.68 (m, 4H), 1.33 (br s, 4H), 1.20 (t,
J = 7.2 Hz, 4H); 13C NMR (DMSO-d6) d 171.2, 170.6, 156.0, 136.7,
128.2, 127.7, 127.5, 65.6, 59.5, 52.5, 52.3, 32.6, 30.7, 24.1, 22.8.
Calcd for C28H36N4O6S2.HCl: C, 53.79; H, 5.97; N, 8.96. Found: C,
53.64; H, 5.89; N, 9.00.
d6) d 13.00 (s, 1H), 7.89 (d, J = 7.2 Hz, 2H), 7.71 (d, J = 7.5 Hz,
2H), 7.75)7.30 (m, 5H), 7.23 (br s, 5H), 5.06)4.94 (m, 2H),
4.46)4.34 (m, 1H), 4.29)4.19 (m, 2H), 3.66 (br s, 2H), 3.32)3.26
(m, 1H), 3.32)3.26 (m, 1H), 3.16)3.05 (m, 2H), 2.88 (dd, J = 14.1,
9.9 Hz, 1H), 1.20 (d, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) d 172.4,
171.4, 168.7, 155.6, 137.6, 136.9, 129.2, 128.3, 128.0, 127.8, 126.2,
198
Chem Biol Drug Des 2012; 80: 194–202

Cysteinoyl- and Cysteine-containing Dipeptidoylbenzotriazoles with Free Sulfhydryl Groups
teinoylbenzotriazoles 7b in a better yield, the SOCl2 method is on
balance more convenient in case of N-Cbz and N-Fmoc free sulfhy-
dryl cysteinoyl-1H-benzotriazoles 7a,c because of the short reaction
time and the easy workup. Novel coupling reagents 7a–c are crys-
talline (mp¢s 155–175 ꢀC), stable at 20 ꢀC in air, and sufficiently
resistant to hydrolysis to be used in partially aqueous solution.
Results and Discussion
Preparation of N-Cbz-, N-Boc-, and N-Fmoc-L-
cysteines 4a–c
The amino group of L-cysteine 2 has been selectively acylated using
N-Cbz-1H-benzotriazole 1a (23,24), N-Boc-1H-benzotriazole 1b
(25,26), and N-Fmoc-1H-benzotriazole 1c. According to our previ-
ously reported method (32), reactions of N-Cbz, N-Boc-, and N-
Fmoc-1H-benzotriazoles 1a–c with L-cysteine 2 at 20 ꢀC in the
presence of 2 equivalents of triethylamine in CH3CN-H2O (3:1) each
gave selectively the corresponding N-protected, free sulfhydryl L-cy-
steines 2a–c (Scheme 1, Table 1) in yields of 70–75%. As previ-
ously discussed (32), such N-acylations probably involve initial
attack by the nucleophilic sulfur anion of L-cysteine at 1a–c; inter-
mediates 3a–c then undergo fast S- to N-acyl transfer to give N-a-
cylcysteines 4a–c (Scheme 1).
Preparation of N-Cbz-, N-Boc-, and N-Fmoc-L-
cysteine-containing di- and tripeptides 8a–h,
(8a + 8a¢)
Scheme 2: Synthesis and peptide coupling reactions of
N-(N¢-Cbz-, N¢-Boc- and N¢-Fmoc-L-Cys)-1H-benzotriazoles 7a–c and
N-(N¢-Cbz- and N¢-Fmoc-a-dipeptidoyl)-1H-benzotriazoles 10a,b.
Reactions of N-Cbz-, N-Boc-, and N-Fmoc-cysteinoyl-1H-benzotriaz-
oles 7a–c (each having free sulfhydryl groups) with various amino
acids and dipeptides in acetonitrile-water (3:1) in the presence of
triethylamine at 20 ꢀC gave the corresponding N-Cbz-, N-Boc-, and
N-Fmoc-L-cysteine dipeptides 8a–e in 73–80% yields and tripep-
tides 8f–h in 75–80% yields having free sulfhydryl groups
(Scheme 2). This procedure allowed the synthesis of dipeptides 8d
(yield 77%) and 8c (yield 73%) containing tryptophan and serine
residues and possessing intact free indole N-H and hydroxyl groups,
respectively (Scheme 2, Table 2). Novel N-Cbz, N-Boc-, and N-Fmoc-
L-cysteine dipeptides 8a–e and tripeptides 8f–h were character-
Scheme 1: Synthesis and S-acylation of N-Cbz-, N-Boc-, and
N-Fmoc-L-cysteine.
S-acylation of N-Cbz-, N-Boc-, and N-Fmoc-L-
cysteines 6a–c
The availability of the free SH group was shown by reacting N-Cbz,
N-Boc-, and N-Fmoc L-cysteines 4a–c with N-acylbenzotriazoles 5
at 20 ꢀC in MeCN-H2O (5:1) for 1 h in the presence of one equiva-
lent KHCO3 to give N-Cbz-, N-Boc-, and N-Fmoc-S-acyl cysteines
6a–c in 78–85% isolated yields (Scheme 1, Table 1).
1
ized by H, 13C NMR spectroscopy and elemental analysis. Dipep-
tides 8a (8a + 8a¢) retained diastereomeric purity, which was
evidenced by Chiral HPLC analysis using a Chirobiotic T column
(detection at 254 nm, flow rate 0.5 mL ⁄ min, and MeOH-H2O (98:2)
as eluent. Thus, diastereomeric mixture (8a + 8a¢) showed two
peaks at 3.99 and 4.77, whereas the single diastereomer 8a
showed one single peak at 4.00, which confirms that there is no
detectable racemization during the reaction.
Preparation of N-(N¢-Cbz-, N¢-Boc-, and N¢-Fmoc-
L-Cys)-1H-benzotriazoles 7a–c
N-Cbz- N-Boc-, and N-Fmoc free sulfhydryl cysteinoylbenzotriazoles
7a–c (Scheme 2, Table 2) were prepared in 20–85% yield by the
reactions of N-protected-L-cysteines 4a–c each with 4 equivalents
of 1H-benzotriazole and 1 equivalent of SOCl2 in THF at 0–10 ꢀC
for 2 h. The very low-yield N-Boc free sulfhydryl cysteinoyl-1H-ben-
zotriazoles 7b (20%) is probably because of the partial deprotection
of N-Boc group in the acidic medium generated by reaction of 1H-
benzotriazole with SOCl2. To improve the yield of 7b, N-Boc-cyste-
ine was treated with 1H-benzotriazole in the presence of DCC to
afford N-Boc free sulfhydryl cysteinoylbenzotriazoles 7b in 80%
yield. Although the DCC method gave N-Boc free sulfhydryl cys-
Preparation of cysteine-containing N-(N¢-Cbz-
and N¢-Fmoc-a-dipeptidoyl)-1H-benzotriazoles
10a,b
N-(N¢-Cbz- and N¢-Fmoc-a-dipeptidoyl)-1H-benzotriazoles 10a,b
(Scheme 2, Table 2) were prepared in 69–81% yield by the reactions
Table 1: Synthesis and S-acylation of N-Cbz-, N-Boc-, and N-Fmoc-L-cysteine
Entry
RCOBt 1a–c
RCOOH 4a–c, Yield (%)a
R1COBt 5
N-protected S-acyl cysteines 6a–c, Yield (%)a
1
2
3
Cbz-Bt 1a
Boc-Bt 1b
Fmoc-Bt 1c
N-Cbz-L-Cys-OH 4a, 75
N-Boc-L-Cys-OH 4b, 71
N-Fmoc-L-Cys-OH 4c, 70
4-NO2-Benzoyl-Bt 5a
4-NO2-Benzoyl-Bt 5a
4-MeO-Benzoyl-Bt 5b
N-Cbz-L-Cys(S-4-NO2-Bz)-OH 6a, 80
N-Boc-L-Cys(S-4-NO2-Bz)-OH 6b, 85
N-Fmoc-L-Cys(S-4-MeO-Bz)-OH 6c,78
aIsolated yield.
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199

Ibrahim et al.
Table 2: Synthesis and peptide coupling reactions of N-(N¢-Cbz-, N¢-Boc-, and N¢-Fmoc-L-Cys)-1H-benzotriazoles 7a,c and N-(N¢-Cbz- and
N¢-Fmoc-a-dipeptidoyl)-1H-benzotriazoles 10a,b
Entry
RCOOBt 7a–c or 10a,b, Yield (%)a
R1-NH2
Peptides 8a–h or 11a–f, Yield (%)a
1
2
N-Cbz-L-Cys-Bt 7a (30), 70
N-Cbz-L-Cys-Bt 7a (30), 70
N-Fmoc-L-Cys-Bt 7c, 85
L-Phe-OH
DL-Phe-OH
L-Phe-OH
N-Cbz-L-Cys-L-Phe-OH 8a (27), 80
N-Cbz-L-Cys-DL-Phe-OH 8a + 8a¢ (27), 80
N-Fmoc-L-Cys-L-Phe-OH 8b, 75
3
4
N-Cbz-L-Cys-Bt 7a (30), 70
N- Fmoc-L-Cys-Bt 7c, 85
L-Ser-OH
L-Trp-OH
N-Cbz-L-Cys-L-Ser-OH 8c, 73
N-Fmoc-L-Cys-L-Trp-OH 8d, 77
5
6
N-Boc-L-Cys-Bt 7b, 80
L-Ala-OH
N-Boc-L-Cys-L-Ala-OH 8e (31), 83
N-Cbz-L-Cys-Gly-Gly-OH 8f, 75
7
N-Cbz-L-Cys-Bt 7a (30), 70
N-Cbz-L-Cys-Bt 7a (30), 70
N-Cbz-L-Cys-Bt 7a (30), 70
N-Cbz-L-Phe-L-Cys-Bt 10a, 81
N-Cbz-L-Phe-L-Cys-Bt 10a, 81
N-Fmoc-Gly-L-Cys-Bt 10b, 69
N-Cbz-L-Phe-L-Cys-Bt 10a, 81
N-Cbz-L-Phe-L-Cys-Bt 10a, 81
N-Cbz-L-Phe-L-Cys-Bt 10a, 81
H-Gly-Gly-OH
H-Leu-Gly-OH
H-Gly-L-Ala-OH
L-Phe-OH
L-Trp-OH
L-Leu-OH
H-Gly-Gly-OH
H-Gly-L-Ala-OH
H-Gly-L-Ala-L-Leu-OH
8
N-Cbz-L-Cys-L-Leu-Gly-OH 8g, 80
N-Cbz-L-Cys-Gly-L-Ala-OH 8h, 80
N-Cbz-L-Phe-L-Cys-L-Phe-OH 11a, 90
N-Cbz-L-Phe-L-Cys-L-Trp-OH 11b, 79
N-Fmoc-L-Phe-L-Cys-L-Leu-OH 11c, 70
N-Cbz-L-Phe-L-Cys-Gly-Gly-OH 11d, 84
N-Cbz-L-Phe-L-Cys-Gly-L-Ala-OH 11e, 80
N-Cbz-L-Phe-L-Cys-Gly-L-Ala-L-Leu-OH 11f, 74
9
10
11
12
13
14
15
aIsolated yield.
of N-Cbz- and N-Fmoc-L-cysteine-containing dipeptides 9a,b pre-
pared from previously reported procedure31 with 4 equivalents of 1H-
benzotriazole and 1 equivalent of SOCl2 in THF at )30 ꢀC for 4 h.
membered ring. Treatment of diamines 12a,b with 2 equivalents
N-(N¢-Cbz-Cys)-1H-benzotriazole 7a in THF-H2O for 3 h, afforded
cyclic disulfide-bridged 14- and 16-membered cyclic peptides via
non-isolable intermediates 13a,b which were oxidized in situ to
give 14a,b in 50% yield. We think that the reasons behind the
low yields of 14a,b are not only the competing thioesterification
reaction, but also the partial solubility of 14a,b in the extraction
solvent (2N HCl) used to remove benzotriazole at the end of the
reaction. (Scheme 3).
Novel coupling reagents 10a,b are crystalline (mp¢s 140–156 ꢀC),
stable at 20 ꢀC in air, and sufficiently resistant to hydrolysis to be
used in partially aqueous solution.
Preparation of N-Cbz- and N-Fmoc- internal
L-cysteine-containing tri-, tetra-, and
pentapeptides 11a–f
Reactions of N-Cbz- and N-Fmoc-dipeptidoyl-1H-benzotriazoles
10a,b (each having free sulfhydryl groups) with amino acids, dipep-
tides, and tripeptide in acetonitrile-water (5:1) in the presence of
triethylamine at 0–5ꢀC gave the corresponding N-Cbz- and N-Fmoc
free sulfhydryl internal L-cysteine-containing tripeptides 11a–c, in
70–90% yields, tetrapeptides 11d,e in 80–84% yields and penta-
peptide 11f in 74% yield having free sulfhydryl groups (Scheme 2,
Table 2). This procedure allowed the synthesis of internal cysteine-
containing tri-, tetra-, and pentapeptides 11a–f having free sulfhy-
dryl groups under mild conditions and in good yields. Novel N-Cbz-
and N-Fmoc-L-cysteine tripeptides 11a–c, tetrapeptides 11d,e and
Scheme 3: Synthesis of cyclic disulfide-bridged (14–16 mem-
bered ring) peptides 14a,b.
1
pentapeptide 11f were characterized by H, 13C NMR spectroscopy,
and elemental analysis.
Conclusion
Synthesis of disulfide-bridged cysteine peptides
Disulfide bridges possess improved stability against enzymatic deg-
radation and play critical roles in stabilizing the tertiary structures
of peptides and proteins (33). Many previous efforts toward the
controlled construction of disulfide bridges have required complex
protection strategies for chain assembly and for selective cleavage
of the sulfhydryl protecting groups (34–37).
In conclusion, we have prepared stable crystalline N-protected, free
sulfhydryl cysteinoylbenzotriazoles 7a–c from N-protected, free sulf-
hydryl cysteines; and N-protected, free sulfhydryl cysteine-containing
dipeptidoylbenzotriazoles from N-protected, free sulfhydryl cysteine-
containing dipeptides. N-Protected, free sulfhydryl cysteinoylbenzo-
triazoles 7a–c formed N-terminal cysteine-containing N-protected,
free sulfhydryl dipeptides and tripeptides selectively under mild
reaction conditions with no detectable racemization in good yields.
Novel N-protected, free sulfhydryl, cysteine-containing dip-
eptidoylbenzotriazoles 10a,b formed internal cysteine-containing N-
We have now prepared cysteine-containing cyclic disulfide-bridged
cyclic peptide 14a with a 14-membered ring and 14b with a 16-
200
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Cysteinoyl- and Cysteine-containing Dipeptidoylbenzotriazoles with Free Sulfhydryl Groups
protected, free sulfhydryl tripeptides, tetrapeptides, and pentapep-
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(2008) Targeting RNA with cysteine-constrained peptides. Bioorg
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sulfhydryl cysteinoylbenzotriazole was used to form 14- and 16-
membered disulfide-bridged cyclic peptides, making them efficient
reagents for peptide synthesis.
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Acknowledgments
We thank the University of Florida, The Kenan Foundation and King
Abdulaziz University, Jeddah, Saudi Arabia for financial support.
Authors thank Dr Nader E. Abo-Dya and Dr C. D. Hall for their help-
ful discussions. Mr T. S. Ibrahim is grateful to the ministry of higher
education in Egypt for a scholarship.
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