Microwave-assisted chemical ligation of S-acyl peptides containing non-terminal cysteine residues

Article abstract of DOI:10.1039/c1ob05740e

An efficient approach for the synthesis of a series of S-acyl peptides containing internal cysteine residues has been developed and the chemical long-range ligation of these S-acyl peptides via 5-, 8-, 11- and 14-membered cyclic transition states has been

Full text of DOI:10.1039/c1ob05740e

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Organic &
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PAPER
Microwave-assisted chemical ligation of S-acyl peptides containing
non-terminal cysteine residues†
Finn K. Hansen,‡a Khanh Ha,‡a Ekaterina Todadze,a Aaron Lillicotch,a Alexander Freya and
Alan R. Katritzky*a,b
Received 11th May 2011, Accepted 29th June 2011
DOI: 10.1039/c1ob05740e
An efficient approach for the synthesis of a series of S-acyl peptides containing internal cysteine
residues has been developed and the chemical long-range ligation of these S-acyl peptides via 5-, 8-, 11-
and 14-membered cyclic transition states has been investigated. Our results include the first examples of
successful isopeptide ligations starting from S-acyl peptides containing non-terminal cysteine residues
and indicate that the cyclic transition states studied in this present paper are decreasingly favored in the
order of their sizes 5ꢀ14>11ꢀ8.
ligation auxiliaries, but unfortunately, removable cysteine mimics
can sterically hinder ligation and difficulties can arise at the stage
of auxiliary removal.6,8
Other strategies to extend the repertoire of native chemical
ligation include (i) conversion of cysteine residues into alanine9–11
and serine;12 (ii) ligation at a b-mercapto-phenylalanine residue13
which is subsequently desulfurized to give phenylalanine; (iii) use
of a g-thiolated valine building block as an alternative precursor
to valine.14
An useful approach for the synthesis of cysteine containing
peptides is isopeptide ligation methodology. Yoshiya et al.15 syn-
thesized S-acyl peptides containing N-terminal cysteine residues;
subsequent S→N intramolecular acyl migration then furnished
native peptide bonds. Very recently, we described the chemical
ligation of selectively S-acylated cysteine peptides to form native
peptides via 11- and 14-membered cyclic transition states.16,17
This method allows the synthesis of native peptides from S-acyl
peptides with a C-terminal cysteine without utilizing auxiliaries.
An extension of our previously reported isopeptide
methodology16,17 utilizing non-terminal cysteine residues could
significantly expand the applicability of this isopeptide ligation
approach. We herein document the first isopeptide ligations to
form native peptides from non-terminal cysteine residues: our
examples involve 11- and 14-membered transition states.
Introduction
Decades of optimization have made stepwise solid-phase peptide
synthesis (SPPS) a routine and reliable tool for preparing peptides
up to around 25 amino acids long;1,3 however, yields generally de-
crease as the length of the polypeptide increases further. Cysteine-
containing peptides are valuable intermediates for the synthesis
of larger peptides and proteins utilizing the chemical ligation of
unprotected peptides.2,4–6 The ligation of an unprotected peptide
thioester in aqueous solution to another unprotected peptide
possessing an N-terminal cysteine with formation of an amide
bond is termed native chemical ligation (NCL).7 The development
of a synthetic strategy for the preparation of a polypeptide by
means of native chemical ligation relies on a suitable cysteine
ligation site. Unfortunately, cysteine is a relatively rare amino acid
(1.3% average content)8 and is not always available in a terminal
position. Moreover, some amino acid-cysteine bonds such as Pro-
Cys, Asp-Cys, Glu-Cys and Lys-Cys can be difficult to access by
chemical ligation.8 Recently, Haase and Seitz reported a chemical
ligation approach utilizing internal cysteine residues to accelerate
thioester-based peptide ligations compared to direct aminolysis
reactions of a cysteine-lacking control peptide.8 However, this
method required relatively long reaction times (48–72 h) and the
ligation products were not isolated.8
The classical NCL method is limited to peptides possessing an
N-terminal cysteine residue. To overcome this requirement of a
specifically placed cysteine residue one approach is the use of thiol
Results and discussion
aCenter for Heterocyclic Compounds, Department of Chemistry, Univer-
sity of Florida, Gainesville, FL 32611-7200, USA. E-mail: katritzky@
chem.ufl.edu; Fax: +1 352-392-9199; Tel: +1 352-292-7022
Study of the feasibility of S→N acyl migration via an 8-membered
compared with a 5-membered cyclic transition state
bDepartment of Chemistry, King Abdulaziz University, Jeddah, 21589, Saudi
Arabia
The protected dipeptide dimer 2 was prepared in 79% yield
by mixed anhydride coupling of bis-Boc-cystine 1 and H-Gly-
OCH3 according to a literature procedure.18 To investigate the
feasibility of chemical ligation through a 5-membered transition
state, the Boc-protected dipeptide dimer 2 was first reacted with
† Electronic supplementary information (ESI) available: General methods,
experimental details for intermediates 2, 3, 4, 7, 8, 14a,b, 15a,b, 21 and 22;
HPLC chromatograms and MS Spectra. See DOI: 10.1039/c1ob05740e
‡ These authors contributed equally to this work.
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tributylphosphine to afford the dipeptide monomer 3 in 55%
yield. Subsequent S-acylation of 3 with Fmoc-Gly-Bt19 (Bt =
benzotriazol-1-yl) furnished the S-acyl peptide 4 in 88% yield.
Next, 4 was Boc-deprotected selectively with methanol saturated
with hydrochloric acid gas. The crude HCl salt 5 was treated
with triethylamine to give the corresponding free base which
as expected underwent classical S→N acyl migration via a 5-
membered transition state to furnish the native tripeptide 6 in
72% yield (Scheme 1).
We then investigated the chemical ligation of 11 via an 8-
membered cyclic transition state. The S-acyl tripeptide 11 was
dissolved in a mixture 0.4 M NaH2PO4/Na2HPO4 buffer (pH =
7.8) and acetonitrile (7 : 1). Acetonitrile was used as co-solvent
to dissolve the starting material. The reaction mixture was
subjected to microwave irradiation for 3 h at 70 ◦C and 50
W (Scheme 2). Interestingly, the HPLC-MS (ESI) analysis of
the reaction mixture revealed that the major component was
unreacted starting material 11. The (+)ESI-MSn spectra of the
major product (m/z 455 [M+H]+ ion) were nearly identical with
those of 11. Moreover, the HPLC-MS (ESI) analysis of a mixture
of 11 and the product from the ligation experiment detected only
one MW 454 compound, whose characteristics matched those of
the S-acyl tripeptide 11. Absence of the S→N acyl migration
11→12 suggests that the necessary S→N acyl migration via an
8-membered cyclic transition state for 11→12 is disfavored.
Study of S→N acyl migration via an 11-membered cyclic
transition state
To study possible chemical ligation via an 11-membered cyclic
transition state, we first prepared the N-Boc-protected dipeptides
14a,b in 65–69% yield by peptide coupling reactions of Boc-
Gly-Bt21 with L-amino acids in partially aqueous solution in
the presence of triethylamine (Scheme 3). Mixed anhydride
couplings of the dipeptide dimer 7 with 2 equiv. of each of
the dipeptides 14a,b furnished the tetrapeptide dimers 15a,b in
72–75% yield (Scheme 3). NMR analysis of 15a,b revealed no
detectable epimerization. Next, the cleavage of the disulfides 15a,b
afforded the monomer tetrapeptides 16a,b (62–66%), which were
subsequently S-acylated with Cbz-L-Ala-Bt in the presence of
triethylamine. The Boc-protected S-acyl tetrapeptides 17a,b were
purified by flash column chromatography or recrystallization and
then isolated in 80–92% yield. Acid-catalyzed deprotection of Boc
group proceeded smoothly to form the desired S-acyl tetrapeptides
18a,b (78–86%, Scheme 3).
Scheme 1 Chemical ligation of S-acyl dipeptide 5.
The preparation of the starting material 11 for a possible S→N
acyl migration via an 8-membered transition state is illustrated
in Scheme 2. First, we deprotected the Boc-protected dipeptide
dimer 2 utilizing saturated hydrochloric acid in methanol to afford
the dipeptide dimer 7 as dihydrochloride (85% yield) followed by
mixed anhydride coupling of 7 with 2 equiv. of Boc-glycine to
furnish the novel tripeptide dimer 8 in 62% yield. Treatment of the
disulfide 8 with tributylphosphine gave the tripeptide monomer
9 (67%, Scheme 2). S-Acylation of 9 with Cbz-L-Ala-Bt20 in the
presence of triethylamine provided the key intermediate 10 (82%
yield) which on deprotection by MeOH–HCl gave the desired S-
acyl tripeptide 11 in 85% yield (Scheme 2).
Scheme 3 Synthesis of S-acyl tetrapeptides 18a,b.
We investigated the S→N acyl migration in compounds 18a,b
via 11-membered cyclic transition states. Our chemical ligation
experiment on 18a was carried out under microwave irradiation at
50 ◦C and 50 W for 1 h. After workup, HPLC-MS (ESI) analysis
Scheme 2 Synthesis and attempted chemical ligation of S-acyl tripeptide
11.
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of the crude ligation mixture showed the presence of two major
products in 70 : 30 ratio (88% combined crude yield22). The most
abundant (+)ESI-MS peak was produced by the intermolecular
transacylation product 20a (MW 772) as shown by (+)ESI-MS
dissociation of its m/z 773 [M+H]+ ion. The HPLC-MS (ESI)
analysis confirmed the second major product to be the desired
ligation product 19a (Scheme 4).
anhydride coupling of 21 with Boc-Gly-L-Phe-OH 15b afforded
the pentapeptide dimer 22 in 74% yield. NMR analysis of 22
revealed no detectable epimerization during the peptide coupling.
Cleavage of the disulfide bond in 22 furnished the monomer 23
(67%) which was S-acylated with Cbz-L-Ala-Bt to provide the
Boc-protected S-acyl pentapeptide 24 (86%). Final deprotection
of the Boc group in 24 afforded the desired starting material 25
(85%, Scheme 5).
Scheme 4 Chemical ligation of S-acyl tetrapeptides 18a,b.
Similarly, the chemical ligation experiment of the S-acyl peptide
18b under the same conditions described above provided the
desired ligation product 19b. HPLC-MS analysis revealed that
this ligation experiment yielded the expected ligation product 19b
and the intermolecular transacylation product 20b in 33 : 67 ratio
(86% combined crude yield,22 Scheme 4).
The product mixture of the ligation experiment of S-acyl
tetrapeptide 18a was subsequently purified by semi-preparative
HPLC, which allowed the isolation of the desired ligation product
19a as well as isolation of the intermolecular transacylation
product 20a in yields of 23% and 52%, respectively. Both products
were characterized by analytical HPLC and HRMS analysis
(Table 1).
Scheme 5 Synthesis of S-acyl pentapeptide 25.
Chemical ligation from S-acyl pentapeptide 25 through a 14-
membered cyclic transition state was investigated by dissolving
25 in 0.4 M phosphate buffer (pH = 7.8) and acetonitrile (7 : 1)
and subjecting the mixture to microwave irradiation (50 ◦C, 50
W, 1h). HPLC-MS (ESI) analysis of the crude ligation mixture
showed the presence of two main products in 57 : 43 ratio (87%
combined crude yield23). The major product of this experiment
was the expected ligation product 26. Again, the intermolecular
transacylation product 27 was formed as side product (Scheme 6).
The subsequent separation of 26 by semi-preparative HPLC
afforded the purified ligation product 26 in 41% yield, which was
characterized by analytical HPLC and HRMS analysis (Table 2).
These are the first examples of successful isopeptide ligations
starting from non-terminal S-acyl peptides and the results strongly
suggest chemical long-range ligation via 11- and 14-membered
cyclic transition states as a promising approach for the synthesis
of native peptides.
The formation of the side products 20 could be explained as
follows: in the first step the intramolecular S→N acyl migration
provides the desired product 19, which is later S-acylated by
another equivalent of the starting material 18 to form the
transacylation product 20 by intermolecular reaction (Scheme 4).
Study of S→N acyl migration via a 14-membered cyclic transition
state
Competitive ligation experiment
The synthesis of starting material 25 for a possible S→N
ligation via a 14-membered transition state was accomplished
in a five-step procedure starting from the tripeptide dimer 8.
After initial deprotection of 8 to form 21, subsequent mixed
We repeated the chemical ligation of S-acyl tetrapeptide 18a in the
presence of 10 equivalents of glycinamide 28 under similar reaction
conditions as described above (50 ◦C, 50 W, 1h). Glycinamide 28
was chosen as model compound to mimic the possible intermolec-
ular reaction at the thioester moiety. However, our HPLC-MS
Table 1 Characterization data of isolated products 19a and 20a
Product
Isolated yield [%]
Purity [%]b
HRMS [M+Na]+ found
Table 2 Characterization data of the isolated ligation product 26
19a
20a
23
> 95
> 95
1155.4423c,d
795.3027e
52a
Product
Isolated yield [%]
41
Purity [%]a
HRMS [M+Na]+ found
1337.4629b,c
a The isolated yield was calculated according to the following equation:
isolated yield = (2 x [20a])/[18] b Purity based on analytical HPLC c Isolated
as disulfide dimer d HRMS calc. [M+Na]+: 1155.4461 e HRMS calc.
[M+Na]+: 795.2994.
26
> 95
a Purity based on analytical HPLC b Isolated as disulfide dimer c HRMS
calc. [M+Na]+: 1337.4578.
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chemical ligation approach, our methodology allows the isolation
of the S-acyl peptide intermediates, which might be useful for
several possible synthetic and biological applications. In order to
optimize the isopeptide ligation we are currently investigating the
S→N acyl migration via various longer and intermediate cyclic
transition states.
Experimental
General procedure for Boc deprotection of peptides 4, 10, 17a,b
and 24 to give the corresponding unprotected peptides 5, 11, 18a,b
and 25. HCl gas was passed through a solution of peptide 4,
10, 17a,b or 24 in methanol (15 mL) for 30 min. The methanol
solution was concentrated under vacuum and diethyl ether (20 mL)
was added. The turbid solution was left to crystallize in the freezer
overnight. The solid formed was filtered and washed with dry ethyl
acetate (10 mL) and diethyl ether (10 mL) and dried to give the
corresponding deprotected peptide 5, 11, 18a,b or 25.
Scheme 6 Chemical ligation experiment of S-acyl pentapeptide 25.
(ESI) analysis did not detect any compound 29, which would
be the product of N-acylation of glycinamide 28. Instead, the
HPLC-MS (ESI) analysis of the crude ligation mixture revealed
that the desired ligation product 19a and the intermolecular
transacylation product 20a were again formed in 30 : 70 ratio
(Scheme 7). The results of this competition experiment further
support the intramolecular nature of our isopeptide ligation.
General procedure for the synthesis of peptides 9, 16a,b and 23.
A mixture of tributylphosphine (0.607 g, 3 mmol) and dimer
peptide 8, 15a,b or 22 (1.5 mmol) in MeOH : water (9 : 1, 20 mL)
was stirred at rt for 2 h under argon. The solvent was evaporated
and the residue was dissolved in diethyl ether (15 mL). The
solution was dried over magnesium sulfate and concentrated under
reduced pressure. The crude peptides were purified according
to the following procedures. Peptide 9 was recrystallized from
diethyl ether : hexanes. Compound 16a was recrystallized from
CH2Cl2 : hexanes. Compounds 16b and 23 were recrystallized from
MeOH : diethyl ether. The precipitates were washed with cold
hexanes (5 mL) and CH2Cl2 (5 mL) and dried under reduced
pressure.
General procedure for the preparation of S-acyl peptides 10,
17a,b and 24. Cbz-L-Ala-Bt (0.325 g, 1 mmol) was added to
a mixture of 9, 16a,b or 23 (1 mmol) and triethylamine (0.1 g,
1 mmol) in acetonitrile (20 mL). The mixture was stirred for 3 h
at rt and the solvent was removed under reduced pressure. The
crude product 17a was purified by flash column chromatography
using ethyl acetate : hexanes (gradient) as eluent. The crude
compounds 10, 17b and 24 were purified according to the following
procedure. The residue was dissolved in ethyl acetate (20 mL),
extracted with 2 N HCl (2 ¥ 20 mL), water (15 mL), and brine
(10 mL). The organic layer was dried over magnesium sulfate
and concentrated under reduced pressure. Compound 10 was
recrystallized from CH2Cl2 : hexanes. The S-acyl peptides 17b
and 24 were recrystallized from ethyl acetate : hexanes. The solids
obtained were filtered, washed with diethyl ether (5 mL) and dried.
Scheme 7 Competitive ligation experiment of 18a in the presence of
glycinamide 28.
Very recently, our group performed a computational investiga-
tion on the intramolecular nature of this ligation approach and
found that hydrogen bonds arising in certain conformations of
S-acyl peptides can provide additional stabilization of the cyclic
transition states.24 These hydrogen bonds clearly favor the 11-
membered over the 8-membered cyclic transition state.24
Conclusions
In summary, we have efficiently and conveniently synthesized
several novel S-acyl peptides containing internal cysteine residues.
The chemical ligation studies of these S-acyl peptides via 5-,
8-, 11- and 14-membered cyclic transition states show that the
8-membered transition state is clearly disfavored, whereas the
11- and 14-membered transition states are relatively favored for
our long-range ligation approach. These results indicate that the
transition states studied in this present paper are decreasingly
favored in the order of their sizes 5ꢀ14>11ꢀ8. Our microwave-
assisted isopeptide ligation offers the following advantages: (i)
short reaction times (1 h) at moderate temperature (50 ◦C), (ii)
chemical ligation from non-terminal cysteine residues and (iii)
avoidance of ligation auxiliaries. In contrast to the classical native
General procedure for chemical ligation of S-acyl peptides 11,
18a,b and 25. The respective S-acyl peptide hydrochloride 11,
18a,b or 25 (0.05 mmol) was suspended in degassed phosphate
buffer (NaH2PO4/Na2HPO4) (0.4 M, pH 7.8, 7 mL) and acetoni-
trile (~1 mL) was added dropwise until the starting material was
dissolved. The mixture was subjected to microwave irradiation
(for compounds 18a,b and 25: 50 ◦C, 50 W, 1 h; for compound
◦
11: 70 C, 50 W, 3 h) under argon. The reaction was allowed to
cool to room temperature, acetonitrile was removed under reduced
pressure and the residue was acidified with 2 N HCl to pH = 1.
The mixture was extracted with ethyl acetate (3 ¥ 20 mL), the
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combined organic extracts were dried over MgSO4 and the solvent
was removed under reduced pressure. The ligation mixture was
weighed and then a solution in methanol (1 mg mL-1) was analyzed
by HPLC-MS. Compounds 19a, 20a and 26 were subsequently iso-
lated by semi-preparative HPLC and characterized by analytical
HPLC and HRMS analysis (see ESI†).
1H), 3.95–4.08 (m, 2H), 4.38 (t, J = 7.2 Hz, 1H), 4.66 (br s, 1H),
5.06–5.20 (m, 2H), 5.43 (d, J = 6.6 Hz, 1H), 5.55 (br s, 1H), 6.89
(d, J = 7.4 Hz, 1H), 7.18 (br s, 1H), 7.36 (br s, 5H); 13C NMR
(75 MHz, CDCl3) d 18.2, 28.5, 29.7, 41.4, 44.5, 52.5, 53.4, 57.2,
67.6, 80.6, 128.3, 128.5, 128.8, 136.1, 156.3, 156.6, 170.0, 170.1,
170.8, 202.5.
(R)-1-(9H-Fluoren-9-yl)-3,6,10,13-tetraoxo-2,14-dioxa-7-thia-
4,11-diazapentadecan-9-aminium chloride (5). White solid, 85%
(5S,9R)-methyl 9-(2-aminoacetamido)-5-methyl-3,6,10-trioxo-
1-phenyl-2-oxa-7-thia-4,11-diazatridecan-13-oate hydrochloride
(11). White microcrystals, 85% yield, mp 130–135 ◦C; Anal.
Calcd for C19H27ClN4O8S·1.5H2O: C 44.06; H 5.84; N 10.82.
Found: C 43.95; H 6.05; N 10.78; 1H NMR (300 MHz, DMSO-d6)
d 1.26 (d, J = 7.0 Hz, 3H), 2.73–2.77 (m, 1H), 3.00–3.06 (m, 1H),
3.62 (br s, 5H), 3.85 (d, J = 5.2 Hz, 2H), 4.20 (t, J = 7.1 Hz, 1H),
4.54–4.56 (m, 1H), 5.02–5.11 (m, 2H), 7.37 (br s, 5H), 8.08–8.10
(m, 1H), 8.15 (br s, 3H), 8.70–8.72 (m, 1H), 8.82 (d, J = 8.5 Hz,
1H); 13C NMR (75 MHz, DMSO-d6) d 17.4, 28.2, 30.3, 40.8, 51.8,
56.7, 65.8, 127.8, 128.0, 128.4, 136.8, 155.9, 166.1, 169.7, 169.9,
201.7.
◦
yield, mp 203–206 C; Anal. Calcd for C23H25N3O6S.HCl·2H2O:
C 50.78; H 5.56; N 7.72. Found: C 50.81; H 5.07; N 7.47; 1H NMR
(300 MHz, DMSO-d6) d 3.29–3.42 (m, 2H), 3.61 (s, 3H), 3.90 (d,
J = 5.4 Hz, 2H), 3.96 (d, J = 5.9 Hz, 2H), 4.08 (br s, 1H), 4.24
(t, J = 6.8 Hz, 1H), 4.34 (d, J = 6.8 Hz, 2H), 7.33 (t, J = 7.4 Hz,
2H), 7.41 (t, J = 7.4 Hz, 2H), 7.71 (d, J = 7.4 Hz, 2H), 7.88 (d,
J = 7.4 Hz, 2H), 8.13 (t, J = 5.9 Hz, 1H), 8.54 (br s, 3H), 9.12
(t, J = 5.4 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) d 28.6, 40.9,
46.7, 50.5, 51.3, 52.0, 66.1, 120.3, 125.3, 127.2, 127.8, 140.8, 143.8,
156.6, 167.3, 169.6, 197.9.
Chemical ligation of Cys-(S-(Fmoc-L-Ala))-Gly-OCH3 hy-
drochloride (5) to form native tripeptide (6). (R)-1-(9H-
Fluoren-9-yl)-3,6,10,13-tetraoxo-2,14-dioxa-7-thia-4,11-diaza-
pentadecan-9-aminium chloride (0.58 g, 1 mmol) was dissolved in
a mixture of water (8 mL) and acetonitrile (24 mL). Triethylamine
(0.168 mL, 1.2 mmol) was added and the mixture was stirred at
room temperature for 1 h under argon. The reaction was acidified
to pH 1 using 2 N HCl and extracted with ethyl acetate (2 ¥ 10 mL).
The ethyl acetate layer was washed with 2 N HCl (3 ¥ 15 mL), sat.
NaCl solution (20 mL) and dried over MgSO4. The ethyl acetate
solution was concentrated under vacuum and hexane was added.
The turbid solution was left to crystallize in the freezer overnight.
The solid formed was filtered and dried to give the corresponding
native tripeptide 6. White microcrystals, 72% yield, mp 88–92 ◦C;
Anal. Calcd for C23H25N3O6S: C 58.59; H 5.34; N 8.91. Found: C
Boc-Gly-L-Leu-L-Cys-Gly-OCH3 (16a). White microcrystals,
66% yield, mp 152–154 ◦C; Anal. Calcd for C19H34N4O7S: C 49.34;
1
H 7.41; N 12.11. Found: C 49.51; H 7.67; N 11.99; H NMR
(300 MHz, DMSO-d6) d 0.79–0.92 (m, 6H), 1.37 (s, 9H), 1.41–1.52
(m, 2H), 1.53–1.67 (m, 1H), 2.29 (t, J = 8.5 Hz, 1H), 2.62–2.89
(m, 2H), 3.55 (d, J = 6.0 Hz, 2 H), 3.62 (s, 3H), 3.76–3.97 (m, 2H),
4.25–4.48 (m, 2H), 6.97 (t, J = 5.7 Hz, 1H), 7.88 (d, J = 8.0 Hz,
1H), 8.11 (d, J = 8.2 Hz, 1H), 8.38 (t, J = 5.8 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6) d 21.6, 23.1, 24.0, 26.1, 28.2, 40.7, 40.8, 43.2,
50.9, 51.7, 54.8, 78.1, 155.7, 169.3, 170.1, 172.0.
Boc-Gly-L-Phe-L-Cys-Gly-OCH3 (16b). White microcrystals,
62% yield, mp 158–162 ◦C; Anal. Calcd for C22H32N4O7S: C 53.21;
1
H 6.50; N 11.28. Found: C 52.83; H 6.43; N 10.91; H NMR
(300 MHz, DMSO-d6) d 1.35 (br s, 9H), 2.31 (t, J = 8.2 Hz, 1H),
2.72–2.84 (m, 3H), 3.01–3.05 (m, 1H), 3.45–3.60 (m, 2H), 3.63
(br s, 3H), 3.87 (br s, 2H), 4.42–4.44 (m, 1H), 4.58 (br s, 1H),
6.90 (br s, 1H), 7.22 (br s, 5H), 7.97 (d, J = 7.6 Hz, 1H), 8.30 (d,
J = 7.6 Hz, 1H), 8.37 (br s, 1H); 13C NMR (75 MHz, DMSO-d6)
d 26.2, 28.2, 37.4, 40.7, 43.1, 51.8, 53.8, 54.9, 78.1, 126.3, 128.0,
129.3, 137.6, 155.7, 169.3, 170.1, 171.1.
1
58.72; H 5.17; N 8.62; H NMR (300 MHz, CDCl3) d 2.89–2.97
(m, 1H), 3.02–3.07 (m, 1H), 3.64 (s, 3H), 3.70 (t, J = 7.3 Hz, 1H),
3.93–9.99 (m, 2H), 4.05–4.09 (m, 2H), 4.21–4.24 (m, 1H), 4.39 (d,
J = 6.9 Hz, 2H), 5.54–5.59 (m, 1H), 6.17 (br s, 1H), 7.13–7.19 (m,
1H), 7.28 (t, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.59 (d, J =
7.4 Hz, 2H), 7.75 (d, J = 7.4 Hz, 2H), 8.47 (br s, 1H); 13C NMR
(75 MHz, DMSO-d6) d 40.8, 43.4, 46.7, 51.8, 62.2, 65.8, 74.9, 78.6,
120.1, 125.3, 127.1, 127.7, 140.7, 143.9, 156.6, 169.4, 170.0, 170.5.
Boc-Gly-L-Leu-L-Cys(S-Cbz-L-Ala)-Gly-OCH3 (17a). White
microcrystals, 80% yield, mp 95–97 ◦C; Anal. Calcd for
C30H45N5O10S: C 53.96; H 6.79; N 10.49. Found: C 53.67; H 6.70;
(R)-Methyl 9-(mercaptomethyl)-2,2-dimethyl-4,7,10-trioxo-3-
oxa-5,8,11-triazatridecan-13-oate (9). White microcrystals, 67%
yield, mp 77–79 ◦C; Anal. Calcd for C13H23N3O6S: C 44.69;
1
N 10.30; H NMR (300 MHz, DMSO-d6) d 0.78–0.92 (m, 6H),
1.26 (d, J = 7.2 Hz, 3 H), 1.37 (s, 9H), 1.40–1.51 (m, 2H), 1.52–
1.67 (m, 1H), 3.02 (dd, J = 13.3, 8.8 Hz, 1H), 3.22 (dd, J = 13.4,
5.3 Hz, 1H), 3.56 (d, J = 6.0 Hz, 2H), 3.62 (s, 3H), 3.78–3.92 (m,
2H), 4.13–4.25 (m, 1H), 4.26–4.44 (m, 2H), 4.99–5.15 (m, 2H),
6.95 (t, J = 5.9 Hz, 1H), 7.24–7.42 (m, 5H), 7.85 (d, J = 8.0 Hz,
1H), 8.07 (d, J = 7.2 Hz, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.31 (t, J =
5.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) d 17.3, 21.6, 23.1,
24.0, 28.1, 30.0, 40.7, 41.0, 43.2, 51.0, 51.7, 56.7, 65.7, 78.1, 127.7,
127.9, 128.3, 136.7, 155.7, 169.3, 169.8, 172.0, 201.9.
1
H 6.63; N 12.03. Found: C 44.29; H 6.77; N 11.67; H NMR
(300 MHz, CDCl3) d 1.45 (s, 9H), 1.85 (dd, J = 11.3, 6.5 Hz,
1H), 2.64–2.72 (m, 1H), 3.3 (br s, 1H), 3.74 (s, 3H), 3.82 (t, J =
5.2 Hz, 2H), 3.87–3.95 (m, 1H), 4.10–4.18 (m, 1H), 4.75–4.79 (m,
1H), 5.28 (br s, 1H), 7.19 (br s, 1H), 7.30 (br s, 1H); 13C NMR
(75 MHz, CDCl3) d 27.1, 28.5, 41.4, 44.6, 52.5, 54.3, 80.6, 156.8,
170.3, 170.7.
(S)-S-((R)-2-(2-((tert-butoxycarbonyl)amino)acetamido)-4-
((methoxycarbonyl)amino)-3-oxobutyl) 2-(((benzyloxy)-carbonyl)-
amino)propanethioate (10). White microcrystals, 82% yield, mp
62–67 ◦C; Anal. Calcd for C24H34N4O9S: C 51.98; H 6.18; N 10.10.
Found: C 51.70; H 5.87; N 9.75; 1H NMR (300 MHz, CDCl3) d
1.44 (s, 12H), 3.37–3.41 (m, 2H), 3.72 (br s, 4H), 3.81–3.88 (m,
Boc-Gly-L-Phe-L-Cys(S-Cbz-L-Ala)-Gly-OCH3 (17b). White
microcrystals, 92% yield, mp 164–167 ◦C; Anal. Calcd for
C33H43N5O10S: C 56.48; H 6.18; N 9.98. Found: C 56.17; H 6.20; N
9.97; 1H NMR (300 MHz, DMSO-d6) d 1.26 (d, J = 6.9 Hz, 3H),
1.35 (s, 9H), 2.77 (dd, J = 13.7, 9.3 Hz, 1H), 3.00–3.06 (m, 2H),
7166 | Org. Biomol. Chem., 2011, 9, 7162–7167
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3.21 (dd, J = 13.2, 5.4 Hz, 1H), 3.63 (br s, 5H), 3.87 (br s, 2H), 4.19
(t, J = 7.3 Hz, 1H), 4.38–4.47 (m, 1H), 4.54 (br s, 1H), 5.01–5.10
(m, 2H), 6.90 (t, J = 5.4 Hz, 1H), 7.17–7.28 (m, 5H), 7.35 (br s,
5H), 7.92 (d, J = 7.7 Hz, 1H), 8.09 (d, J = 7.3 Hz, 1H), 8.38 (t, J =
5.1 Hz, 1H), 8.45 (d, J = 7.8 Hz, 1H); 13C NMR (75 MHz, DMSO-
d6) d 18.6, 28.9, 30.9, 38.8, 42.1, 44.7, 52.8, 54.0, 56.2, 58.4, 68.0,
80.9, 128.0, 128.9, 129.2, 129.6, 129.7, 130.5, 138.1, 138.3, 158.4,
171.5, 172.2, 172.6, 173.4, 203.6.
Calcd for C30H39ClN6O9S: C 51.83; H 5.65; N 12.09. Found: C
51.78; H 5.76; N 12.26; H NMR (300 MHz, CD3OD) d 1.40
1
(d, J = 7.1 Hz, 3H), 2.98–3.06 (m, 1H), 3.24–3.28 (m, 2H), 3.43–
3.50 (m, 1H), 3.66–3.91 (m, 5H), 3.98–4.06 (m, 4H), 4.35 (t, J =
6.9 Hz, 1H), 4.60–4.67 (m, 2H), 5.14 (s, 2H), 7.29–7.39 (m, 10H);
13C NMR (75 MHz, CD3OD) d 18.0, 31.0, 38.3, 41.8, 42.1, 43.7,
52.9, 53.9, 57.0, 58.4, 68.0, 128.0, 128.9, 129.2, 12.6, 129.7, 130.4,
138.2, 138.4, 158.5, 167.9, 171.6, 171.7, 172.5, 173.8, 203.7.
H-Gly-L-Leu-L-Cys(S-Cbz-L-Ala)-Gly-OCH3
hydrochloride
Acknowledgements
(18a). White microcrystals, 78% yield, mp 82–86 ◦C; Anal.
Calcd for C25H38ClN5O8S: C 49.70; H 6.34; N 11.59. Found:
C 49.52; H 6.41; N 11.70; H NMR (300 MHz, DMSO-d6) d
We thank the University of Florida, the Kenan Foundation and
King Abdulaziz University, Jeddah, Saudi Arabia for financial
support. We thank Dr C. D. Hall for helpful discussions, Dr Jodie
Johnson for his help in HLPC-MS analyses, Dr M. C. A. Dancel
for her help with HRMS analyses and the referees for helpful and
constructive comments.
1
0.80–0.94 (m, 6H), 1.26 (d, J = 7.3 Hz, 3H), 1.40–1.54 (m, 2H),
1.54–1.71 (m, 1H), 3.06 (dd, J = 13.2, 8.6 Hz, 1H), 3.22 (dd, J =
13.3, 5.7 Hz, 1H), 3.59 (s, 2H), 3.62 (s, 3H), 3.84 (d, J = 5.8 Hz,
2H), 4.11–4.25 (m, 1H), 4.32–4.46 (m, 2H), 4.99–5.12 (m, 2H),
7.26–7.42 (m, 5H), 8.09 (d, J = 7.3 Hz, 1H), 8.21 (br s, 3H), 8.41
(t, J = 5.7 Hz, 1H), 8.47 (d, J = 8.2 Hz, 1H), 8.65 (d, J = 8.0 Hz,
1H); 13C NMR (75 MHz, DMSO-d6) d 17.4, 21.5, 23.1, 24.1,
30.0, 40.8, 41.0, 51.3, 51.7, 56.7, 65.8, 127.8, 127.9, 128.4, 136.7,
155.8, 165.9, 169.9, 169.9, 171.6, 201.9.
Notes and references
1 In our peptide nomenclature the prefixes di-, tri-, tetra- etc refer to the
number of amino acid residues in the main peptide chain; amino acid
residues attached to sulfur are designated as S-acyl peptides.
2 The term isopeptide ligation is used for non-native chemical ligations
from S-acyl peptides to furnish native peptides in a stepwise approach.
A cysteine containing peptide is converted to a S-acyl peptide,
deprotected and then transferred to a native peptide by a subsequent
S→N acyl migration. In contrast to the native chemical ligation this
isopeptide methodology allows the isolation and characterization of
the S-acyl peptides and to migrate the S-acyl group in an independent
step.
3 B. Merrifield, Science, 1986, 232, 341.
4 S. Quesnel and J. R. Silvius, Biochemistry, 1994, 33, 13340.
5 I. Photaki, J. Am. Chem. Soc., 1966, 88, 2292.
6 C. Marinzi, S. J. Bark, J. Offer and P. E. Dawson, Bioorg. Med. Chem.,
2001, 9, 2323.
7 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science,
1994, 266, 776.
8 C. Haase and O. Seitz, Eur. J. Org. Chem., 2009, 2096.
9 L. Z. Yan and P. E. Dawson, J. Am. Chem. Soc., 2001, 123, 526.
10 B. L. Pentelute and S. B. H. Kent, Org. Lett., 2007, 9, 687.
11 Q. Wan and S. J. Danishefsky, Angew. Chem., Int. Ed., 2007, 46, 9248.
12 R. Okamoto and Y. Kajihara, Angew. Chem., Int. Ed., 2008, 47, 5402.
13 D. Crich and A. Banerjee, J. Am. Chem. Soc., 2007, 129, 10064.
14 J. Chen, Q. Wan, Y. Yuan, J. Zhu and S. J. Danishefsky, Angew. Chem.,
Int. Ed., 2008, 47, 8521.
15 T. Yoshiya, N. Ito, T. Kimura and Y. Kiso, J. Pept. Sci., 2008, 14, 1203.
16 A. R. Katritzky, N. E. Abo-Dya, S. R. Tala and Z. K. Abdel-Samii,
Org. Biomol. Chem., 2010, 8, 2316.
17 A. R. Katritzky, S. R. Tala, N. E. Abo-Dya, T. S. Ibrahim, S. A. El-Feky,
K. Gyanda and K. M. Pandya, J. Org. Chem., 2011, 76, 85.
18 S. A. Khan and B. W. Erickson, J. Am. Chem. Soc., 1981, 103, 7374.
19 A. R. Katritzky, A. Singh, D. N. Haase and M. Yoshioka, Arkivoc,
2009, 47.
H-Gly-L-Phe-L-Cys(S-Cbz-L-Ala)-Gly-OCH3
hydrochloride
(18b). White microcrystals, 86% yield, mp 172–175 ◦C; Anal.
Calcd for C28H36ClN5O8S·2H2O: C 49.88; H 5.38; N 10.39. Found:
C 50.06; H 5.70; N 10.76; 1H NMR (300 MHz, DMSO-d6) d 1.27
(d, J = 7.0 Hz, 3H), 2.72–2.80 (m, 1H), 3.07–3.11 (m, 2H), 3.23
(dd, J = 13.0, 5.7 Hz, 1H), 3.62 (br s, 5H), 3.86 (d, J = 5.1 Hz, 2H),
4.20 (t, J = 7.2 Hz, 1H), 4.40–4.44 (m, 1H), 4.61–4.64 (m, 1H),
5.05 (dd, J = 17.1, 12.4 Hz, 2H), 7.14–7.44 (m, 10 H), 8.11 (d, J =
7.3 Hz, 1H), 8.18 (br s, 3H), 8.49 (br s, 1H), 8.71 (d, J = 7.9 Hz,
1H), 8.79 (d, J = 8.0 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) d
17.4, 30.1, 37.8, 40.8, 51.8, 54.3, 56.7, 65.8, 126.4, 127.8, 127.9,
128.1, 128.4, 129.3, 136.7, 137.5, 155.8, 165.7, 169.9, 170.7, 201.9.
Boc-Gly-L-Phe-Gly-L-Cys-Gly-OCH3 (23). White microcrys-
◦
tals, 67% yield, mp 188–193 C; Anal. Calcd for C24H35N5O8S1:
1
C 52.07; H 6.37; N 12.65. Found: C 52.00; H 6.62; N 11.79; H
NMR (300 MHz, DMSO-d6) d 1.35 (br s, 9H), 2.34 (br s, 1H),
2.68–2.83 (m, 3H), 3.00–3.05 (m, 1H), 3.62 (br s, 5H), 3.72–4.00
(m, 4H), 4.46–4.50 (m, 2H), 6.88 (br s, H), 7.23 (br s, 5H), 8.07
(d, J = 5.6 Hz, 2H), 8.41 (br s, 1H), 8.52 (br s, 1H); 13C NMR
(75 MHz, CD3OD) d 27.1, 28.9, 38.2, 42.1, 43.9, 44.7, 52.8, 56.8,
57.2, 81.0, 128.0, 129.7, 130.4, 138.4, 158.7, 171.8, 172.8, 174.5.
Boc-Gly-L-Phe-Gly-L-Cys(S-Cbz-L-Ala)-Gly-OCH3
(24).
White microcrystals, 86% yield, mp 126–128 ◦C; Anal. Calcd
for C35H46N6O11S: C 55.40; H 6.11; N 11.07. Found: C 55.02; H
20 A. R. Katritzky, K. Suzuki and S. K. Singh, Synthesis, 2004, 16, 2645.
21 E. Masiukiewicz, B. Rzeszotarska, I. Wawrzycka-Gorczyca and E.
Kolodziejczyk, Synth. Commun., 2007, 37, 1917.
1
6.05; N 11.00; H NMR (300 MHz, CD3OD) d (ppm) 1.36 (d,
J = 7.3 Hz, 3H), 1.43 (s, 9H), 2.96 (dd, J = 13.9, 8.7 Hz, 1H),
3.17–3.24 (m, 2H), 3.43 (dd, J = 13.9, 5.1 Hz, 1H), 3.60–3.69 (m,
1H), 3.70 (br s, 5H), 3.97 (br s, 3H), 4.29 (q, J = 7.3 Hz, 1H),
4.56–4.66 (m, 2H), 5.11 (s, 2H), 7.19–7.36 (m, 10H); 13C NMR
(75 MHz, CD3OD) d (ppm) 18.0, 28.9, 31.0, 38.4, 42.2, 43.8, 44.7,
52.8, 54.0, 56.5, 58.4, 68.0, 81.0, 127.9, 128.9, 129.2, 129.7, 130.4,
138.2, 138.5, 158.5, 171.6, 172.5, 172.8, 174.2, 203.4.
22 The combined crude yield of 19 and 20 was calculated based on
the isolated amount of the product mixture 19:20 according to the
following equation: combined crude yield = ([19] + 2 x [20])/[18]. The
S-deacylated tetrapeptide side products were removed during the work-
up.
23 The combined crude yield of 26 and 27 was calculated based on
the isolated amount of the product mixture 26:27 according to the
following equation: combined crude yield = ([26] + 2 x [27])/[25]. The
S-deacylated pentapeptide side product was removed during the work-
up.
H-Gly-L-Phe-L-Gly-Cys(S-Cbz-L-Ala)-Gly-OCH3 hydrochlo-
ride (25). White microcrystals, 85% yield, mp 131–134 ◦C; Anal.
24 A. A. Oliferenko and A. R. Katritzky, Org. Biomol. Chem., 2011, 9,
4756.
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The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 7162–7167 | 7167
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