Solution-phase synthesis of chiral N-, O-, and S-acyl isopeptides

Article abstract of DOI:10.1055/s-0033-1340089

A convenient synthesis of chiral N-, O-, and S-acyl monoiso- and diisopeptides from di- and tripeptides containing tryptophan, tyrosine, and cysteine units using benzotriazole is reported in solution phase. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0033-1340089

PAPER
▌67
paper
Solution-Phase Synthesis of Chiral N-, O-, and S-Acyl Isopeptides
Synthesis of Chiral N-, O- and S-Acyl Isopeptides
Sumaira Liaqat,a,b Siva S. Panda,a Abdul Rauf,b Abdulrahman O. Al-Youbi,c Alan R. Katritzky*a,c
a
Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
b
Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
c
Chemistry Department, King Abdulaziz University, Jeddah 21589, Saudi Arabia
Fax +1(352)3929199; E-mail: katritzky@chem.ufl.edu
Received: 25.09.2013; Accepted after revision: 11.10.2013
tions of biologically relevant peptides and peptide conju-
gates in high yields and purity, under mild reaction
conditions, with full retention of the original chirality.23,24
Abstract: A convenient synthesis of chiral N-, O-, and S-acyl mo-
noiso- and diisopeptides from di- and tripeptides containing trypto-
phan, tyrosine, and cysteine units using benzotriazole is reported in
solution phase.
N-(Pg-α-aminoacyl)benzotriazoles 1a–e and N-(Pg-di-
Key words: amino acids, peptides, chirality, benzotriazole method, peptidoyl)benzotriazoles 1f–h were prepared following
reported procedures25 and were reacted with tryptophan,
solution-phase synthesis
tryrosine, and cysteine to obtain the corresponding di- and
tripeptides. These were reacted further with N-(Pg-α-ami-
noacyl)benzotriazoles and N-(Pg-dipeptidoyl)benzotri-
azoles to obtain mono- and diisotripeptides and -tetrapeptides.
Solid-phase peptide synthesis (SPPS) has been used rou-
tinely for the synthesis of peptides and proteins. However,
the synthesis of ‘difficult sequence’ containing peptides is
still a challenge in peptide chemistry since these peptides
are often obtained in low yield and purity by SPPS.1–3 The
difficult sequences are generally hydrophobic and prone
to aggregation in solvent during chain elongation and final
purification. This is attributed to inter/intramolecular hy-
drophobic interactions and hydrogen-bond networks
formed among resin-bound peptide chains, resulting in the
formation of extended secondary structures such as β-
sheets.
Synthesis of Tryptophan Isopeptides
Benzotriazolides 1a–c were coupled with free tryptophan
(2) at 0 to 20 °C in the presence of triethylamine in aceto-
nitrile to give Cbz-protected dipeptides 3a–c (Table 1).
These dipeptides 3a–c were N-acylated by (Cbz-protect-
ed-α-aminoacyl)benzotriazoles 1a,b,d in the presence of a
base (Et3N, DIPEA, K2CO3, or DBU) in acetonitrile to ob-
tain protected monoisotripeptides 4a–d (Table 2). DBU
gave better results than the other evaluated bases (Scheme 1).
Kiso and co-workers reported that 21% D-Val was detect-
ed during the synthesis of Boc-Thr(Fmoc-Val) via solid
phase, while epimerization was completely avoided in the
solution phase.4 In addition, due to the presence of an ad-
ditional amino group, N-, O-, or S-acyl isopeptides are
generally hydrophilic, which is advantageous in effective
purification by HPLC. The native peptides are then gener-
ated from the corresponding N-, O, or S-acyl isopeptide
via an N-to-N,5 O-to-N,6 or S-to-N7–9 intramolecular acyl
migration reaction. The strategy facilitates the synthesis
of peptides with ‘difficult sequences’. The O-acyl isopep-
tide method has already been used in various fields in-
cluding peptide synthesis,4a,10–14 ‘click peptide’ (‘switch
peptide’) concept,15–18 macromolecules,19 peptide local-
ization,20 protein splicing,21 and proteomics.22
Table 1 Preparation of N-Protected Dipeptides 3a–c Containing a
Tryptophan Unit
Product 3
Yield (%)
79
Mp (°C)
Lit. mp (°C)
Z-Gly-L-Trp-OH, 3a
139–141
154–155
183–185
142–14326
154–15527
185–18727
Z-L-Ala-L-Trp-OH, 3b 78
Z-L-Val-L-Trp-OH, 3c 77
Table 2 Preparation of N-Acyl Monoisotripeptides 4a–d
Product 4
Yield (%)
Mp (°C)
We now report the efficient single-step preparation of chi-
ral N-, O, or S-acyl isopeptides incorporating tryptophan,
Z-Gly-L-Trp(Z-L-Phe)-OH, 4a
79
78
75
76
48–50
56–58
54–56
52–54
tyrosine, and cysteine. N-Acylbenzotriazoles are advanta- Z-L-Ala-L-Trp(Z-L-Phe)-OH, 4b
geous for N-, O-, C-, and S- acylation,23 especially where
Z-L-Val-L-Trp(Z-Gly)-OH, 4c
the corresponding acid chlorides are unstable or prone to
racemization. N-[Protected (Pg)-α-aminoacyl]- and N- Z-L-Val-L-Trp(Z-L-Ala)-OH, 4d
(Pg-dipeptidoyl)benzotriazoles have enabled fast prepara-
SYNTHESIS 2014, 46, 0067–0072
Advanced online publication: 26.11.2013
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1
1
4
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DOI: 10.1055/s-0033-1340089; Art ID: SS-2013-M0645-OP
© Georg Thieme Verlag Stuttgart · New York

68
S. Liaqat et al.
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(Table 3, Scheme 3). These di- and tripeptides 9a–f were
S-acylated by N-(Pg-α-aminoacyl)benzotriazoles and
dipeptidoylbenzotriazoles in the presence of potassium
bicarbonate to obtain protected monoisotri-, -tetra-, and
-pentapeptides 10a–h (Table 4, Scheme 3).
R1
NH
Et3N, MeCN
Cbz
Bt
+
NH
OH
N
H
O
O
OH
H
H2N
N
Cbz
N
O
H
Table 3 Preparation of N-Protected Peptides 9a–f Containing a Cys-
teine Unit
R1
O
1a–c
2
3a–c
Product 9
Yield (%)
Mp (°C)
R1
Z-L-Ala-L-Cys-OH, 9a
96
96
98
90
92
95
170–171
169–170
125–126
156–158
177–179
170–172
DBU, MeCN
Cbz
Bt
N
Z-L-Val-L-Cys-OH, 9b
H
O
1a,b,d
Z-L-Phe-L-Cys-OH, 9c
Z-L-Phe-Gly-L-Cys-OH, 9d
Z-L-Phe-L-Ala-L-Cys-OH, 9e
Z-L-Ala-L-Phe-L-Cys-OH, 9f
O
H
N
Cbz
N
R1
O
H
OH
N
Cbz
N
H
R1
O
Table 4 Preparation of S-Acyl Peptides 10a–h
Product 10
4a–d
Yield (%) Mp (°C)
Scheme 1 Preparation of N-acyl monoisotripeptides 4a–d
Z-L-Phe-L-Cys(Z-Gly)-OH, 10a
45a
171–173
145–147
142–143
167–169
161–163
169–171
170–171
146–148
Synthesis of Tyrosine Isopeptide
Z-L-Val-L-Cys(Z-Gly)-OH, 10b
84
86
97
95
94
96
The benzotriazolide 1e was coupled with free tyrosine (5)
at 0 to 20 °C in the presence of DBU in DMF to give Boc-
protected dipeptide 6. The dipeptide 6 was O-acylated by
N-(Pg-α-aminoacyl)benzotriazole 1b in the presence of
triethylamine to obtain the protected monoisotripeptide 7
(Scheme 2).
Z-L-Ala-L-Cys(Z-L-Phe)-OH, 10c
Z-L-Ala-L-Phe-L-Cys(Z-L-Ala)-OH, 10d
Z-L-Ala-L-Cys(Z-L-Ala-L-Phe)-OH, 10e
Z-L-Phe-Gly-L-Cys(Z-L-Ala)-OH, 10f
Z-L-Phe-L-Ala-L-Cys(Z-L-Ala-)-OH, 10g
Synthesis of Cysteine Isopeptides
The benzotriazolides 1b–h were coupled with free cyste-
ine (8) at 0 to 20 °C in the presence of triethylamine in
acetonitrile to give N-protected di- and tripeptides 9a–f
Z-L-Phe-Gly-L-Cys(Z-L-Ala-L-Phe)-OH, 10h 98
a Compound was isolated by extraction with EtOAc.
OH
OH
O
H
+
DBU, DMF
OH
N
Boc
OH
Bt
Boc
N
H
H2N
N
H
O
O
O
1e
5
6, 68%
Cbz
O
Z-Ala-Bt (1b)
Et3N, MeCN
NH
O
O
H
N
N
H
OH
Boc
O
7, 65%
Scheme 2 Preparation of O-acyl monoisotripeptide 7
Synthesis 2014, 46, 67–72
© Georg Thieme Verlag Stuttgart · New York

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Synthesis of Chiral N-, O- and S-Acyl Isopeptides
69
n = 0,1
Pg
HN
R2
O
H
R2
Pg
N
Bt
N
R2
O
O
H
R1
O
SH
O
SH
O
H
NH
Pg
N
N
R2
O
R1
n = 0,1
+
Et3N, MeCN
Bt
H
OH
Pg
OH
H
N
O
R1
N
N
O
H2N
H
H
KHCO3, MeCN
Pg
O
R1
S
n = 0,1
R2
O
8
H
n = 0,1
1b–h
OH
N
N
N
H
H
9a–f
O
R1
O
n = 0,1
10a–h
Scheme 3 Preparation of O-acyl monoisotripeptides 10a–h
13C NMR (DMSO-d6): δ = 174.2, 169.9, 157.4, 138.0, 137.0, 129.3,
128.7, 128.2, 124.6, 121.9, 119.4, 119.1, 112.3, 110.6, 66.4, 53.9,
44.2, 28.1.
In summary, N-peptidoylbenzotriazoles are advantageous
coupling reagents that (i) are sufficiently reactive to form
amide bonds at ambient temperature; (ii) are stable
enough to resist side reactions and can be stored in the
crystalline state at room temperature; (iii) provide good
yields without detectable racemization; (iv) are almost al-
ways crystalline; (v) are relatively insensitive to moisture
and can be used in aqueous solution, and (vi) are inexpen-
Anal. Calcd for C21H21N3O5: C, 63.79; H, 5.35; N, 10.63. Found: C,
63.71; H, 5.226; N, 10.37.
Dipeptides 3b,c gave also physical and spectral data in conformity
with the reported values.26,27
Protected Monoisotripeptides 4a–d; General Procedure
sive to prepare. Hence N-(Pg-α-aminoacyl)benzotriazole To a precooled solution of tryptophan containing the appropriate
peptide 3a–c (0.5 mmol) in MeCN (10 mL) and Et3N (1.5 equiv) at
and N-(Pg-α-dipeptidoyl)benzotriazole reagents allow ef-
ficient peptide couplings to generate monoisopeptides via
N-, O-, and S-acylation.
0 °C was added a solution of N-(Pg-α-aminoacyl)benzotriazole
1a,b, or d (0.5 mmol) in MeCN (3 mL). After completion of the re-
action (8 h), the reaction mixture was acidified with aq 1 M HCl and
then extracted with EtOAc (10 mL). The organic layer was washed
with H2O (10 mL) and dried (Na2SO4). Evaporation of the solvent
gave the desired product 4a–d, respectively, which was recrystal-
lized from EtOAc–hexanes (Table 2).
Commercial reagents were purchased from Sigma-Aldrich and
were used without purification. Solvents were purified by distilla-
tion. Melting points were determined on a capillary point apparatus
equipped with a digital thermometer. NMR spectra were recorded
1-{[(Benzyloxy)carbonyl]-L-phenylalanyl}-Nα-{[(benzyl-
in CDCl3 or CD3OD on Mercury or Gemini NMR spectrometers op-
oxy)carbonyl]glycyl}-L-tryptophan (4a)
erating at 300 MHz for 1H (with TMS as an internal standard) and
Yield: 0.52 g (79%); off-white solid; mp 48–50 °C.
75 MHz for 13C. Elemental analyses were performed on a Carlo
Erba-EA1108 instrument. Analytical TLC was performed on E.
Merck silica gel 60 F254 plates and visualized by UV and KMnO4
1H NMR (DMSO-d6): δ = 12.70 (br s, 1 H), 10.87 (s, 1 H), 8.09 (d,
J = 7.8 Hz, 1 H), 7.66 (d, J = 8.6 Hz, 1 H), 7.54 (d, J = 7.8 Hz, 1 H),
7.49–7.24 (m, 16 H), 7.12–6.94 (m, 3 H), 5.04–4.80 (m, 4 H), 4.58–
staining. Flash column chromatography was performed on E.
4.46 (m, 1 H), 4.28–4.14 (m, 1 H), 3.75–3.55 (m, 2 H), 3.27–2.98
Merck silica gel 60 (40–63 mm). Yields refer to chromatographical-
(m, 4 H).
ly and spectroscopically pure compounds. Mass spectrometry was
13C NMR (DMSO-d6): δ = 173.3, 173.2, 169.0, 156.5, 156.0, 137.9,
137.1, 137.0, 136.1, 129.1, 128.3, 128.3, 128.3, 128.2, 127.8, 127.7,
127.6, 127.5, 127.2, 123.7, 120.9, 118.4, 118.2, 111.4, 109.6, 65.5,
65.3, 55.5, 52.9, 43.3, 36.5, 27.2.
HRMS (–ESI-TOF): m/z [M – H]– calcd for C38H36N4O8: 675.2460;
found: 675.2477.
done with electrospray ionization (ESI).
Cbz-Protected Dipeptides 3a–c; General Procedure
To the respective N-(Pg-α-aminoacyl)benzotriazole 1a–c (0.5
mmol) in MeCN (10 mL) was added a solution of tryptophan (2;
102 mg, 0.5 mmol) and Et3N (0.5 mL) in H2O (3 mL). The reaction
mixture was stirred for 8 h at 0 °C. The mixture was acidified by aq
1 M HCl and extracted with EtOAc (10 mL). The organic layer was
washed with aq 1 M HCl (3 mL) and brine (5 mL), and dried
(MgSO4). After evaporation of solvent, the residue was triturated
with Et2O and the solid formed was filtered and dried under vacuum
to give dipeptides 3a–c, respectively (Table 1).
Nα-{[(Benzyloxy)carbonyl]-L-alanyl}-1-{[(benzyloxy)carbonyl]-
L-phenylalanyl}-L-tryptophan (4b)
Yield: 0.53 g (78%); off-white solid; mp 56–58 °C.
1H NMR (DMSO-d6): δ = 12.69 (s, 1 H), 10.86 (s, 1 H), 8.04 (d,
J = 7.7 Hz, 1 H), 7.65 (d, J = 8.4 Hz, 1 H), 7.53 (d, J = 7.9 Hz, 1 H),
7.39–7.19 (m, 18 H), 7.06 (t, J = 7.5 Hz, 1 H), 6.98 (t, J = 7.4 Hz, 1
H), 5.08–4.90 (m, 4 H), 4.49 (q, J = 7.0 Hz, 1 H), 4.28–4.04 (m, 2
H), 3.23–3.02 (m, 3 H), 2.91–2.71 (m, 1 H), 1.21 (t, J = 6.8 Hz, 3
H).
13C NMR (DMSO-d6): δ = 173.3, 173.2, 172.5, 156.0, 155.6, 137.9,
137.0, 136.0, 129.1, 128.3, 128.3, 128.2, 128.2, 127.8, 127.7, 127.6,
127.5, 127.2, 126.4, 123.7, 120.9, 118.4, 118.2, 111.3, 109.6, 65.4,
65.3, 55.5, 52.9, 49.9, 39.5, 36.5, 27.0, 18.2.
[(Benzyloxy)carbonyl]glycyl-L-tryptophan (3a)
Yield: 0.3 g (79%); white solid; mp 139–141 °C (Lit.26 mp 142–143 °C).
1H NMR (DMSO-d6): δ = 12.65 (br s, 1 H), 10.87 (d, J = 2.4 Hz, 1
H), 8.08 (d, J = 7.7 Hz, 1 H), 7.53 (d, J = 7.8 Hz, 1 H), 7.48–7.22
(m, 7 H), 7.14 (s, 1 H), 7.07 (t, J = 7.5 Hz, 1 H), 6.99 (t, J = 7.4 Hz,
1 H), 5.03 (s, 2 H), 4.74–4.28 (m, 1 H), 3.77–3.52 (m, 2 H), 3.18
(dd, J = 14.7, 5.2 Hz, 1 H), 3.05 (dd, J = 14.6, 7.8 Hz, 1 H).
© Georg Thieme Verlag Stuttgart · New York
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70
S. Liaqat et al.
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HRMS (–ESI-TOF): m/z [M – H]– calcd for C39H38N4O8: 689.2617;
found: 689.2637.
organic layer was washed with aq 4 M HCl (3 mL) and brine (5
mL), and dried (Na2SO4). After evaporation of the solvent, the res-
idue was triturated with Et2O–hexanes (1:1) and the solid formed
was filtered and dried under vacuum to give the respective dipep-
tides 9a–c and tripeptides 9d–f (Table 3).
Nα-{[(Benzyloxy)carbonyl]-L-valyl}-1-{[(benzyloxy)carbon-
yl]glycyl}-L-tryptophan (4c)
Yield: 0.46 g (75%); off-white solid; mp 54–56 °C.
1H NMR (DMSO-d6): δ = 12.59 (br s, 1 H), 10.85 (s, 1 H), 8.15 (d,
J = 7.5 Hz, 1 H), 7.53 (d, J = 7.8 Hz, 1 H), 7.38–7.22 (m, 12 H),
7.20–7.16 (m, 1 H), 7.06 (t, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.4 Hz, 1
H), 5.12–4.95 (m, 4 H), 4.49 (dd, J = 13.8, 7.3 Hz, 2 H), 4.09–3.54
(m, 2 H), 3.22–2.80 (m, 2 H), 1.95 (dd, J = 14.1, 7.4 Hz, 1 H), 0.82
(t, J = 7.2 Hz, 6 H).
13C NMR (DMSO-d6): δ = 173.2, 171.2, 159.4, 156.1, 137.1, 136.1,
128.4, 128.3, 127.8, 127.7, 123.6, 120.9, 118.4, 111.3, 109.6, 65.4,
59.9, 52.9, 30.5, 27.1, 19.2, 18.1.
[(Benzyloxy)carbonyl]-L-alanyl-L-cysteine (9a)
Yield: 0.31 g (97%); white solid; mp 170–171 °C.
1H NMR (DMSO-d6): δ = 8.67 (d, J = 7.9 Hz, 1 H), 7.92–7.81 (m,
1 H), 7.81–7.65 (m, 5 H), 5.52–5.35 (m, 2 H), 4.99–4.86 (m, 1 H),
4.61–4.45 (m, 1 H), 3.65–3.33 (m, 2 H), 1.65 (d, J = 7.1 Hz, 3 H).
13C NMR (DMSO-d6): δ = 173.1, 172.2, 156.1, 137.4, 128.8, 128.2,
128.2, 65.9, 51.9, 50.4, 31.1, 18.7.
Anal. Calcd for C14H18N2O5S: C, 51.52; H, 5.56; N, 8.58. Found: C,
51.83; H, 5.55; N,9.10.
HRMS (–ESI-TOF): m/z [M – H]– calcd for C34H36N4O8: 627.2460;
found: 627.2488.
[(Benzyloxy)carbonyl]-L-valyl-L-cysteine (9b)
Yield: 0.17 g (96%); white solid; mp 169–170 °C.
1-{[(Benzyloxy)carbonyl]-L-alanyl}-Nα-{[(benzyloxy)carbonyl]-
L-valyl}-L-tryptophan (4d)
Yield: 0.48 g (76%); off-white solid; mp 52–54 °C.
1H NMR (DMSO-d6): δ =12.84 (s, 1 H), 8.16 (d, J = 7.7 Hz, 1 H),
7.45–7.20 (m, 6 H), 5.04 (s, 2 H), 4.57–4.27 (m, 1 H), 3.94 (dd,
J = 8.9, 6.8 Hz, 1 H), 2.92–2.71 (m, 2 H), 2.43 (t, J = 8.5 Hz, 1 H),
2.09–1.86 (m, 1 H), 0.89 (d, J = 6.7 Hz, 3 H), 0.85 (d, J = 6.6 Hz, 3
H).
13C NMR (DMSO-d6): δ = 171.4, 156.1, 137.1, 128.3, 127.8, 127.6,
65.4, 60.0, 54.3, 30.3, 25.5, 19.2, 18.1.
1H NMR (DMSO-d6): δ = 8.15 (s, 1 H), 7.85–7.72 (m, 2 H), 7.57
(dd, J = 10.7, 7.3 Hz, 1 H), 7.45–7.19 (m, 12 H), 7.09–6.92 (m, 2
H), 5.10–4.90 (m, 4 H), 4.78 (t, J = 6.4 Hz, 1 H), 4.27–4.12 (m, 1
H), 4.04 (dd, J = 7.2, 3.7 Hz, 1 H), 3.47–3.09 (m, 2 H), 2.10–1.90
(m, 1 H), 1.36 (t, J = 7.1 Hz, 3 H), 0.88 (d, J = 7.4 Hz, 3 H), 0.84 (d,
J = 6.2 Hz, 3 H).
13C NMR (DMSO-d6): δ = 176.5, 174.9, 174.8, 173.9, 158.2, 138.1,
138.0, 137.8, 129.6, 129.5, 129.0, 128.8, 128.8, 127.1, 124.8, 122.5,
120.0, 119.4, 115.7, 112.5, 67.5, 61.9, 54.5, 50.8, 32.1, 28.6, 19.8,
18.8, 18.0.
Anal. Calcd for C16H22N2O5S: C, 54.22; H, 6.26; N, 7.90. Found: C,
54.26; H, 6.37; N, 7.82.
[(Benzyloxy)carbonyl]-L-phenylalanyl-L-cysteine (9c)
Yield: 0.39 g (98%); white solid; mp 125–126 °C.
1H NMR (DMSO-d6): δ = 12.95 (br s, 1 H), 8.48 (d, J = 7.7 Hz, 1
H), 7.48 (d, J = 8.8 Hz, 1 H), 7.38–7.10 (m, 10 H), 5.00–4.82 (m, 2
H), 4.61–4.47 (m, 1 H), 4.39–4.24 (m, 1 H), 3.21 (dd, J = 13.7, 4.7
Hz, 1 H), 3.12–2.95 (m, 2 H), 2.74 (dd, J = 13.8, 10.9 Hz, 1 H).
HRMS (–ESI-TOF): m/z [M – H]– calcd for C35H38N4O8: 641.2617;
found: 641.2626.
(S)-3-(4-{[((Benzyloxy)carbonyl)-L-alanyl]oxy}phenyl)-2-{(S)-
2-[(tert-butoxycarbonyl)amino]-3-phenylpropanamido}propa-
noic Acid (7)
13C NMR (DMSO-d6): δ = 171.8, 171.8, 155.8, 138.1, 137.0, 129.2,
128.3, 128.0, 127.7, 127.4, 126.3, 65.2, 56.0, 51.6, 37.5.
To Boc-Phe-Bt (1e; 183 mg, 0.5 mmol) in MeCN (10 mL) was add-
ed a solution of tyrosine (5; 91 mg, 0.5 mmol) and DBU (1.0 mmol)
in DMF (5 mL). The reaction mixture was stirred for 6 h at 20 °C.
The mixture was acidified with aq 2 M HCl and extracted with
EtOAc (10 mL). The organic layer was washed with aq 2 M HCl (3
mL) and brine (5 mL), and dried (MgSO4). The crude product 6 was
treated with Z-Ala-Bt (1b; 109 mg, 0.34 mmol) in the presence of
Et3N (1.5 equiv) in MeCN–H2O (7 mL:3 mL) at 0 °C. After com-
pletion of the reaction (6 h), the mixture was acidified with aq 4 M
HCl. The solution was then extracted with EtOAc (10 mL), the
EtOAc layer was washed with H2O (10 mL), and dried (Na2SO4).
Evaporation of the solvent gave the desired product 7; yield: 0.41 g
(65%); white solid; mp 171–173 °C.
1H NMR (DMSO-d6): δ = 8.12 (d, J = 8.0 Hz, 1 H), 7.95 (d, J = 6.9
Hz, 1 H), 7.40–7.15 (m, 13 H), 6.98 (d, J = 8.3 Hz, 2 H), 6.88 (d,
J = 8.6 Hz, 1 H), 5.07 (s, 2 H), 4.48 (s, 1 H), 4.32 (s, 1 H), 4.18 (s,
1 H), 3.13–2.89 (m, 3 H), 2.69 (t, J = 12.3 Hz, 1 H), 1.42 (d, J = 7.0
Hz, 3 H), 1.28 (s, 9 H).
Anal. Calcd for C20H22N2O5S: C, 59.69, H, 5.51; N, 6.96. Found:
C,60.10; H,5.50; N,6.83.
[(Benzyloxy)carbonyl]-L-phenylalanylglycyl-L-cysteine (9d)
Yield: 0.4 g (90%); white solid; mp 156–158 °C.
1H NMR (DMSO-d6): δ = 12.90 (s, 1 H), 8.39 (t, J = 5.8 Hz, 1 H),
8.07 (d, J = 7.9 Hz, 1 H), 7.58 (d, J = 8.5 Hz, 1 H), 7.40–7.10 (m,
10 H), 4.97 (d, J = 12.9 Hz, 1 H), 4.92 (d, J = 11.9 Hz, 1 H) 4.53–
4.40 (m, 1 H), 4.36–4.21 (m, 1 H), 3.90–3.71 (m, 2 H), 3.04 (dd,
J = 14.0, 4.0 Hz, 1 H), 2.95–2.72 (m, 2 H), 2.43 (t, J = 8.5 Hz, 1 H),
1.36 (s, 1 H).
13C NMR (DMSO-d6): δ = 172.0, 171.7, 168.8, 156.0, 138.2, 137.0,
129.2, 128.3, 128.1, 127.7, 127.5, 126.3, 65.3, 56.2, 54.3, 42.0 37.3,
25.7.
Anal. Calcd for C22H25N3O6S: C, 57.50; H, 5.48; N, 9.14. Found: C,
57.28; H, 5.48; N, 9.00.
[(Benzyloxy)carbonyl]-L-phenylalanyl-L-alanyl-L-cysteine (9e)
Yield: 0.43 g (92%); white solid; mp 177–179 °C.
13C NMR (DMSO-d6): δ = 172.7, 171.8, 171.7, 156.0, 155.2, 149.1,
138.2, 136.9, 135.1, 130.4, 129.2, 128.4, 128.0, 127.9, 127.8, 126.2,
121.2, 78.1, 65.6, 55.8, 53.3, 49.6, 37.4, 36.0, 28.1, 16.8.
HRMS (–ESI-TOF): m/z [M – H]– for C34H39N3O9: 632.2614;
found: 632.2599.
1H NMR (DMSO-d6): δ = 12.93 (br s, 1 H), 8.21 (d, J = 7.9 Hz, 1
H), 7.97 (d, J = 8.9 Hz, 1 H), 7.43 (d, J = 7.5 Hz, 1 H), 7.39–7.16
(m, 10 H), 5.04 (d, J = 12.6 Hz, 1 H), 4.98 (d, J = 12.4 Hz, 1 H),
4.56 (dd, J = 9.1, 4.6 Hz, 1 H), 4.43 (dd, J = 7.0, 4.4 Hz, 1 H), 4.06–
3.94 (m, 1 H), 3.07 (dd, J = 13.6, 4.1 Hz, 1 H), 2.94–2.72 (m, 3 H),
1.19 (dd, J = 15.6, 8.3 Hz, 1 H), 1.12 (d, J = 7.1 Hz, 3 H).
13C NMR (DMSO-d6): δ = 172.3, 171.3, 170.9, 155.7, 137.6, 136.9,
129.3, 128.3, 128.0, 127.8, 127.7, 126.2, 65.4, 54.4, 53.5, 50.2,
37.2, 25.5, 18.0.
N-Protected Di- and Tripeptides 9a–f; General Procedure
To the corresponding N-protected aminoacyl- and dipeptidoylben-
zotriazole 1b–h (0.5 mmol) in MeCN (10 mL) was added a solution
of cysteine (8; 61 mg, 0.5 mmol) and Et3N (0.5 mL) in H2O (3 mL).
The reaction mixture was stirred for 4 h at 0 °C. The mixture was
acidified with aq 4 M HCl and extracted with EtOAc (10 mL). The
Synthesis 2014, 46, 67–72
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of Chiral N-, O- and S-Acyl Isopeptides
71
Anal. Calcd for C23H27N3O6S: C, 58.34; H, 5.75; N, 8.87. Found: C,
57.96; H, 5.81; N, 8.90.
13C NMR (DMSO-d6): δ = 200.6, 172.6, 171.6, 156.0, 155.6, 137.4,
137.0, 136.8, 129.2, 128.4, 128.3, 128.2, 127.8, 127.8, 127.4, 126.5,
65.6, 65.5, 62.7, 51.5, 50.0, 36.5, 29.7, 18.3.
[(Benzyloxy)carbonyl]-L-alanyl-L-phenylalanyl-L-cysteine (9f)
Yield: 0.44 g (95%); white solid; mp 170–172 °C.
Anal. Calcd for C31H33N3O8S: C, 61.27; H, 5.47; N, 6.91. Found: C,
60.88; H, 5.35; N, 7.20.
1H NMR (DMSO-d6): δ = 12.93 (s, 1 H), 8.22 (d, J = 7.9 Hz, 1 H),
7.97 (d, J = 8.9 Hz, 1 H), 7.43 (d, J = 7.4 Hz, 1 H), 7.40–7.15 (m,
10 H), 5.04 (d, J = 12.6 Hz, 1 H), 4.98 (d, J = 12.4 Hz, 1 H), 4.56
(dd, J = 9.2, 4.6 Hz, 1 H), 4.43 (dd, J = 7.1, 4.4 Hz, 1 H), 4.06–3.95
(m, 1 H), 3.07 (dd, J = 13.8, 4.2 Hz, 1 H), 2.92–2.73 (m, 2 H), 2.44
(d, J = 8.7 Hz, 1 H), 1.12 (d, J = 7.2 Hz, 3 H).
13C NMR (DMSO-d6): δ = 172.3, 171.3, 170.9, 155.7, 137.6, 136.9,
129.3, 128.3, 128.0, 127.8, 127.7, 126.2, 65.4, 54.4, 53.5, 50.2,
37.2, 25.5, 18.0.
S-{[(Benzyloxy)carbonyl]-L-alanyl}-N-{[(benzyloxy)carbonyl]-
L-alanyl-L-phenylalanyl}-L-cysteine (10d)
Yield: 0.65 g (97%); white solid; mp 173–175 °C.
1H NMR (DMSO-d6): δ = 12.92 (s, 1 H), 8.60 (d, J = 7.9 Hz, 1 H),
8.20 (d, J = 8.0 Hz, 1 H), 7.42–7.15 (m, 17 H), 5.14–4.88 (m, 4 H),
4.58 (s, 1 H), 4.42–4.24 (m, 1 H), 4.20–3.95 (m, 2 H), 3.29 (s, 1 H),
3.19–3.00 (m, 2 H), 2.88 (dd, J = 14.1, 10.1 Hz, 1 H), 1.21 (d,
J = 6.9 Hz, 3 H), 1.19 (d, J = 6.6 Hz, 3 H).
13C NMR (DMSO-d6): δ = 199.8, 172.8, 172.5, 171.5, 155.6, 155.5,
137.1, 137.0, 136.9, 129.1, 128.3, 128.2, 128.1, 127.8, 127.7, 126.5,
65.4, 65.4, 60.4, 51.5, 49.9, 36.5, 29.4, 18.3, 17.9.
Anal. Calcd for C23H27N3O6S: C, 58.34, H, 5.75; N, 8.87. Found: C,
57.96; H, 5.81; N, 8.90.
S-Acyl Peptides 10a–h; General Procedure
Anal. Calcd for C34H38N4O9S: C, 60.16; H, 5.64; N, 8.25. Found: C,
59.88; H, 5.66; N, 8.21.
To a precooled solution of cysteine containing the appropriate pep-
tide 9a–f (0.5 mmol) in MeCN–H2O (7 mL:3 mL) at 0 °C was added
a solution of N-acylbenzotriazole or N-(Pg-α-aminoacyl)benzotri-
azole 1b–h (0.5 mmol) in MeCN (3 mL) with stirring followed by
addition of KHCO3 (0.14 g) for 10 min in four installments. After
additional stirring for 2–3 h at 0 to 10 °C, the reaction mixture was
acidified with aq 4 M HCl. The solution was then extracted with
EtOAc (10 mL), the EtOAc layer was washed with H2O (10 mL),
and dried (Na2SO4). Evaporation of the solvent gave the respective
desired product 10a–h, which was recrystallized from EtOAc–
hexanes (Table 4).
N-{[(Benzyloxy)carbonyl]-L-alanyl}-S-[(benzyloxy)carbonyl]-
L-alanyl-L-phenylalanyl-L-cysteine (10e)
Yield: 0.64 g (95%); white solid; mp 161–163 °C.
1H NMR (DMSO-d6): δ = 12.92 (s, 1 H), 8.60 (d, J = 7.9 Hz, 1 H),
8.20 (d, J = 8.0 Hz, 1 H), 7.44–7.15 (m, 17 H), 5.10–4.91 (m, 4 H),
4.61–4.58 (m, 1 H), 4.38–4.26 (m, 1 H), 4.16–3.99 (m, 2 H), 3.32–
3.29 (m, 1 H), 3.16–3.02 (m, 2 H), 2.88 (dd, J = 14.1, 10.1 Hz, 1 H),
1.21 (d, J = 6.9 Hz, 3 H), 1.19 (d, J = 6.3 Hz, 3 H).
13C NMR (DMSO-d6): δ = 199.8, 172.8, 172.5, 171.5, 155.6, 155.5,
137.0, 129.1, 128.3, 128.2, 128.2, 127.8, 127.7, 126.5, 65.4, 60.4,
51.5, 49.9, 36.5, 29.4, 18.3, 17.9.
N-{[(Benzyloxy)carbonyl]-L-phenylalanyl}-S-{[(benzyloxy)car-
bonyl]glycyl}-L-cysteine (10a)
Yield: 0.3 g (45%); white solid; mp 171–173 °C.
Anal. Calcd for C34H38N4O9S: C, 60.16; H, 5.64; N, 8.25. Found: C,
60.23; H, 5.82; N, 8.31.
1H NMR (DMSO-d6): δ = 13.00 (s, 1 H), 8.48 (d, J = 6.6 Hz, 1 H),
8.03 (t, J = 5.6 Hz, 1 H), 7.49 (d, J = 8.9 Hz, 1 H), 7.41–7.13 (m, 15
H), 5.14–4.82 (m, 4 H), 4.38–4.29 (m, 2 H), 3.95 (d, J = 6.1 Hz, 2
H), 3.27–2.91 (m, 3 H), 2.77–2.68 (m, 1 H).
13C NMR (DMSO-d6): δ = 198.4, 171.7, 171.4, 156.5, 155.8, 138.1,
137.0, 136.7, 129.2, 128.5, 128.4, 128.3, 128.0, 127.9, 127.7, 127.6,
127.4, 65.8, 65.2, 56.0, 51.7, 50.4, 37.5, 29.1.
S-{[(Benzyloxy)carbonyl]-L-alanyl}-N-[(benzyloxy)carbonyl]-
L-phenylalanylglycyl-L-cysteine (10f)
Yield: 0.61 g (94%); white solid; mp 169–171 °C.
1H NMR (DMSO-d6): δ = 12.95 (s, 1 H), 8.34–8.19 (m, 2 H), 8.05
(d, J = 7.4 Hz, 1 H), 7.52 (d, J = 8.5 Hz, 1 H), 7.40–7.14 (m, 15 H),
5.11–4.84 (m, 4 H), 4.45–4.12 (m, 3 H), 3.83–3.63 (m, 2 H), 3.30–
3.23 (m, 1 H), 3.12–2.96 (m, 2 H), 2.73 (dd, J = 13.8, 10.7 Hz, 1 H),
1.24 (d, J = 7.2 Hz, 3 H).
HRMS (–ESI-TOF): m/z [M – H]– calcd for C30H31N3O8S :
592.1759; found: 592.1746.
13C NMR (DMSO-d6): δ = 201.6, 171.8, 171.4, 168.7, 155.9, 155.8,
138.2, 137.0, 129.2, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.4,
126.2, 65.8, 65.2, 56.7, 56.2, 51.5, 41.7, 37.4, 29.5, 17.3.
N-{[(Benzyloxy)carbonyl]-L-valyl}-S-{[(benzyloxy)carbon-
yl]glycyl}-L-cysteine (10b)
Yield: 0.45 g (84%); white solid; mp 145–147 °C.
1H NMR (DMSO-d6): δ = 8.32 (d, J = 7.8 Hz, 1 H), 7.99 (t, J = 6.1
Hz, 1 H), 7.46–7.19 (m, 11 H), 5.14–4.96 (m, 4 H), 4.40–4.22 (m, 1
H), 3.94 (d, J = 6.4 Hz, 3 H), 3.34 (dd, J = 13.5, 5.3 Hz, 2 H), 3.10
(dd, J = 13.5, 8.4 Hz, 1 H), 2.02–1.95 (m, 1 H), 0.87(d, J = 6 Hz, 3
H), 0.83 (d, J = 6 Hz, 3 H).
13C NMR (DMSO-d6): δ = 198.4, 171.5, 171.3, 156.5, 156.1, 137.1,
136.8, 128.4, 128.4, 127.9, 127.8, 127.6, 65.9, 65.5, 59.9, 51.7,
50.4, 30.6, 29.1, 19.2, 17.9.
Anal. Calcd for C33H36N4O9S: C, 59.63; H, 5.46; N, 8.43. Found: C,
59.24; H, 5.55; N, 8.42.
S-{[(Benzyloxy)carbonyl]-L-alanyl}-N-[(benzyloxy)carbonyl]-
L-phenylalanyl-L-alanyl-L-cysteine (10g)
Yield: 0.66 g (96%); white solid; mp 170–171 °C.
1H NMR (DMSO-d6): δ = 12.92 (br s, 1 H), 8.60 (d, J = 7.9 Hz, 1
H), 8.20 (d, J = 8.1 Hz, 1 H), 7.48–7.16 (m, 17 H), 5.09–4.92 (m, 4
H), 4.65–4.52 (m, 1 H), 4.38–4.26 (m, 1 H), 4.16–4.00 (m, 2 H),
3.31–3.29 (m, 1 H), 3.16–3.02 (m, 2 H), 2.88 (dd, J = 14.1, 10.1 Hz,
1 H) 1.21 (d, J = 6.8 Hz, 3 H), 1.19 (d, J = 6.6 Hz, 3 H).
Anal. Calcd for C26H31N3O8S: C, 57.24; H, 5.73; N, 7.70. Found: C,
57.0; H, 5.78; N, 7.68.
13C NMR (DMSO-d6): δ = 199.8, 172.8, 172.5, 171.5, 155.6, 155.5,
137.0, 129.1, 128.3, 128.2, 128.2, 127.8, 127.7, 126.5, 65.4, 65.4,
60.4, 49.9, 49.8, 36.5, 29.4, 18.3, 17.9.
N-{[(Benzyloxy)carbonyl)]-L-alanyl}-S-{[(benzyloxy)carbon-
yl]-L-phenylalanyl}-L-cysteine (10c)
Yield: 0.51 g (86%); white solid; mp 142–143 °C.
1H NMR (DMSO-d6): δ = 8.24 (d, J = 8.1 Hz, 1 H), 8.15 (d, J = 7.1
Hz, 1 H), 7.44 (d, J = 8.3 Hz, 1 H), 7.37–7.20 (m, 15 H), 5.08–4.94
(m, 4 H), 4.43–4.34 (m, 2 H), 4.17–4.06 (m, 1 H), 3.36 (dd, J = 13.6,
5.5 Hz, 1 H), 3.18–3.05 (m, 2 H), 2.81 (dd, J = 13.9, 11.1 Hz, 1 H),
1.24 (d, J = 7.1 Hz, 3 H).
Anal. Calcd for C34H38N4O9S: C, 60.16; H, 5.64; N, 8.25. Found: C,
60.35; H, 5.67; N, 8.19.
S-[(Benzyloxy)carbonyl]-L-alanyl-L-phenylalanyl-N-[(benzyl-
oxy)carbonyl]-L-phenylalanylglycyl-L-cysteine (10h)
Yield: 0.79 g (98%); white solid; mp 146–148 °C.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 67–72

72
S. Liaqat et al.
PAPER
(8) Ha, K.; Chahar, M.; Monbaliu, J.-C. M.; Todadze, E.;
1H NMR (DMSO-d6): δ = 8.64 (d, J = 8.0 Hz, 1 H), 8.32 (t, J = 5.7
Hz, 1 H), 8.22 (d, J = 8.0 Hz, 1 H), 7.56 (d, J = 8.7 Hz, 1 H), 7.42–
7.15 (m, 21 H), 5.07–4.88 (m, 4 H), 4.65–4.55 (m, 1 H), 4.42–4.26
(m, 2 H), 4.16–4.04 (m, 1 H), 3.85–3.72 (m, 2 H), 3.40–3.29 (m, 1
H), 3.16–3.03 (m, 3 H), 2.96–2.84 (m, 1 H), 2.77 (dd, J = 13.1, 10.0
Hz, 1 H), 1.21 (d, J = 7.2 Hz, 3 H).
13C NMR (DMSO-d6): δ = 199.9, 172.8, 171.4, 168.6, 155.9, 137.1,
137.0, 129.2, 129.1, 128.3, 128.3, 128.2, 128.0, 127.8, 127.7, 127.4,
126.2, 65.2, 60.4, 56.2, 51.7, 49.9, 41.7, 36.5, 29.8, 17.9.
Hansen, F. K.; Oliferenko, A. A.; Ocampo, C. E.; Leino, D.;
Lillicotch, A.; Stevens, C. V.; Katritzky, A. R. J. Org. Chem.
2012, 77, 2637.
(9) Katritzky, A. R.; Tala, S. R.; Dya, N. E. A.; Ibrahim, T. S.;
E-Feky, S. A.; Gyanda, K.; Pandya, K. M. J. Org. Chem.
2011, 76, 85.
(10) Coin, I.; Dolling, R.; Krause, E.; Bienert, M.; Beyermann,
M.; Sferdean, C. D.; Carpino, L. A. J. Org. Chem. 2006, 71,
6171.
(11) Taniguchi, A.; Yoshiya, T.; Abe, N.; Fukao, F.; Sohma, Y.;
Kimura, T.; Hayashi, Y.; Kiso, Y. J. Pept. Sci. 2007, 13, 868.
(12) Lecaillon, J.; Gilles, P.; Subra, G.; Martinez, J.; Amblard, M.
Tetrahedron Lett. 2008, 49, 4674.
(13) Yoshiya, T.; Kawashima, H.; Sohma, Y.; Kimura, T.; Kiso,
Y. Org. Biomol. Chem. 2009, 7, 2894.
(14) Tailhades, J.; Gidel, M.-A.; Grossi, B.; Lecaillon, J.; Brunel,
L.; Subra, G.; Martinez, J.; Amblard, M. Angew. Chem. Int.
Ed. 2010, 49, 117.
(15) Taniguchi, A.; Sohma, Y.; Kimura, M.; Okada, T.; Ikeda, K.;
Hayashi, Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.; Kiso, Y.
J. Am. Chem. Soc. 2006, 128, 696.
HRMS (–ESI-TOF): m/z [M – H]– calcd for C42H45N5O10S:
810.2814; found: 810.2822.
Acknowledgment
We thank the University of Florida and the Kenan Foundation for
financial support. This paper was also funded in part by generous
support from King Abdulaziz University, under grant No. D-
006/431. The authors, therefore, acknowledge the technical and fi-
nancial support of KAU. We also thank Dr. C. D. Hall for useful
suggestions and English checking.
(16) Taniguchi, A.; Sohma, Y.; Hirayama, Y.; Mukai, H.;
Kimura, T.; Hayashi, Y.; Matsuzaki, K.; Kiso, Y.
ChemBioChem 2009, 10, 710.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnuIomprifgtooatrinSugpIoi
n
nfomartit
(17) Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Santos,
S. D.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.;
Saucede, L.; Tuchscherer, G. Angew. Chem. Int. Ed. 2004,
43, 4172.
(18) Kiewitz, S. D.; Kakizawa, T.; Kiso, Y.; Cabrele, C. J. Pept.
Sci. 2008, 14, 1209.
(19) Hentschel, J.; Krause, E.; Borner, H. G. J. Am. Chem. Soc.
2006, 128, 7722.
(20) Akira, S.; Daisuke, T.; Naomi, N.; Shugo, T.; Kohji, I.;
Akira, O. ChemBioChem 2007, 8, 1929.
(21) Perello, M. V.; Hori, Y.; Ribo, M.; Muir, T. W. Angew.
Chem. Int. Ed. 2008, 47, 7764.
(22) Boussert, S.; Perez, I. D.; Kogan, M. J.; Oliveira, E.; Giralt,
E. ACS Nano 2009, 3, 3091.
(23) Bajaj, K.; Panda, S. S.; Nachef, C. E.; Katritzky, A. R. Chem.
Biol. Drug Des. 2012, 80, 17.
(24) Panda, S. S.; Bajaj, K.; Meyers, M. J.; Sverdrup, F. M.;
Katritzky, A. R. Org. Biomol. Chem. 2012, 10, 8985.
(25) Katritzky, A. R.; Angrish, P.; Todadze, E. Synlett 2009,
2392.
References
(1) Tam, J. P.; Lu, Y.-A. J. Am. Chem. Soc. 1995, 117, 12058.
(2) Guichou, J.-F.; Patiny, L.; Mutter, M. Tetrahedron Lett.
2002, 43, 4389.
(3) Alexander, I.; Raimo, F.; Antje, R.; Tatjana, A.; Lothar, J.
Eur. Pat. Appl EP 2487183 A1 20120815, 2012; Chem.
Abstr. 2012, 157, 349369.
(4) (a) Yoshiya, T.; Taniguchi, A.; Sohma, Y.; Fukao, F.;
Nakamura, S.; Abe, N.; Ito, N.; Skwarczynski, M.; Kimura,
T.; Hayashi, Y.; Kiso, Y. Org. Biomol. Chem. 2007, 5, 1720.
(b) Sohma, Y.; Taniguchi, A.; Skwarczynski, M.; Yoshiya,
T.; Fukao, F.; Kimura, T.; Hayashi, Y.; Kiso, Y.
Tetrahedron Lett. 2006, 47, 3013.
(5) Popov, V.; Panda, S. S.; Katritzky, A. R. Org. Biomol.
Chem. 2013, 11, 1594.
(6) Popov, V.; Panda, S. S.; Katritzky, A. R. J. Org. Chem.
2013, 78, 7455.
(7) Panda, S. S.; El-Nachef, C.; Bajaj, K.; Youbi, A. O.;
Oliferenko, A. A.; Katritzky, A. R. Chem. Biol. Drug. Des.
2012, 80, 821.
(26) Anderson, G. W. French patent FR 1406785 A, 1965; Chem.
Abstr. 1965, 63, 72439.
(27) Katritzky, A. R.; Suzuki, K.; Singh, S. K. Synthesis 2004,
2645.
Synthesis 2014, 46, 67–72
© Georg Thieme Verlag Stuttgart · New York