Ligations from tyrosine isopeptides via 12- to 19-membered cyclic transition states

Article abstract of DOI:10.1021/jo4009468

Efficient syntheses of O-acyl Tyr-peptides allow chemical long-range ligation (O-acyl to N-acyl transfer) via each of 12- to 19-membered cyclic transition states. The results represent the first examples of successful isopeptide ligations starting from O-

Full text of DOI:10.1021/jo4009468

Article
pubs.acs.org/joc
Ligations from Tyrosine Isopeptides via 12- to 19-Membered Cyclic
Transition States
Vadim Popov,‡ Siva S. Panda,‡ and Alan R. Katritzky*,‡,§
‡Center for Heterocyclic Compounds, University of Florida, Department of Chemistry, Gainesville, Florida 32611-7200, United
States
§Department of Chemistry, King Abdulaziz University, Jeddah 21589, Saudi Arabia
S
* Supporting Information
ABSTRACT: Efficient syntheses of O-acyl Tyr-peptides allow
chemical long-range ligation (O-acyl to N-acyl transfer) via
each of 12- to 19-membered cyclic transition states. The
results represent the first examples of successful isopeptide
ligations starting from O-acyl Tyr-peptides.
native peptide by O → N intramolecular acyl migration via a 5-
membered transition state.21
INTRODUCTION
■
Synthetic methods for peptides are of great interest: native
chemical ligation (NCL), first developed by Wieland1 and
developed by Kent,2,3 is a chemo- and regioselective reaction of a
peptide-thioester with an N-terminal Cys-peptide that produces
a long chain polypeptide with a native amide bond at the ligation
site through a rapid S- to N-acyl transfer within the initial
thioester.
Alternative approaches to bypass the requirement in classical
NCL of a N-terminal Cys-residue have included (i) traceless
Staudinger ligation,4,5 (ii) native chemical ligation with Phe- and
Val-analogues bearing a sulfhydryl group at the β-position
followed by removal of that group,6−8 (iii) NCL followed by the
conversion of penicillamine to Val,7 (iv) sugar-assisted
ligation,9−11 (v) Cys free “direct aminolysis” methods,12 and
(vi) desulfurization with the formation of Ala-peptide and
protein analogues.13 However, new ligation strategies are still of
significant interest for the synthesis of underivatized and post-
translationally modified peptides and proteins.
Our group developed ligations of S-acylated Cys-peptides22−24
and N-acylated Trp-peptides25 to form native peptides through
various transition states. Recently we demonstrated that such
classic O- to N-acyl shifts via a 5-membered transition state in O-
acyl Ser- and O-acyl Thr-peptides can be extended to 8-
membered and 11-membered transition states. Thus, “traceless”
chemical ligation involving O- to N-acyl shift (at a Ser site)
involving neither Cys nor an auxiliary group at the ligation site is
possible.26
We now report migrations of acyl groups from O-acylated Tyr-
isopeptides (free of Cys) via 12- to 19-membered cyclic
transition states to give natural peptides.
RESULTS AND DISCUSSION
■
We synthesized the monoisotripeptides 5a−c as starting
materials to study the possibility of diverse O- to N-acyl
migrations via 12- to 19-membered cyclic transition states.
Compounds 5a−c were used for ligation studies via 12-, 13- and
14-membered cyclic transition states and after classical coupling
of 5a−c with α-, β- or γ-amino acids provided the starting
monoisotetrapeptides 8a−e for ligation studies via 15- to 19-
membered cyclic transition states. In order to enhance migration
rates, we used exclusively Gly-, β-Ala- and GABA-units in these
monoisopeptide intermediates.
While of great importance, NCL has limitations including (i)
the need of a N-terminal Cys-residue at the ligation site to afford
a peptide containing an internal Cys and (ii) the low abundance
of Cys in globular proteins (1.7% of the residues).4,14,15
Considerable effort has been devoted to the development of
thiol auxiliary groups to overcome the limitation of low
abundance of Cys, but subsequent ligations were found (i)
difficult to complete because of steric hindrance15−19 and (ii)
problematic since extraneous groups in the ligated product may
be difficult to remove.15−19 Another approach involves the
conversion of a Cys-residue into a Ser-residue after NCL,15 but
this requires post-NCL modifications.
Preparation of the Monoisotripeptides 5a−c. Benzo-
triazolides 1a−c of Boc-protected Gly, β-Ala and GABA were
coupled with free Tyr 2 at 20 °C in the presence of base to give
the corresponding Boc-protected dipeptides 3a−c (69−84%)
(Scheme 1). Dipeptides 3a−c were O-acylated by Z-L-Ala-Bt 1d
in the presence of TEA to provide N-protected monoisotripep-
tides 4a−c (71−84%), which after deprotection by HCl solution
in 1,4-dioxane yielded the free monoisotripeptides 5a−c (96−
Kiso et al.20 reported that O-acyl residues within a backbone
significantly altered the secondary structures of native peptides
so that “O-acyl isopeptides” are more hydrophilic and easier to
purify by HPLC than their corresponding native peptides; N-
terminal Ser-isopeptides can rapidly generate the corresponding
Received: May 1, 2013
© XXXX American Chemical Society
A
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Scheme 1. Synthesis of the Monoisotripeptides 5a−c
Scheme 2. Acyl Migration of O-Acyl Isotripeptides 5a−c
Table 1. Chemical Ligation of O-Acyl Isotripeptides 5a−c
ab
,
relative area (%)
product characterization by HPLC−MS
ligated peptide (LP) bis-acylated product (BA)
react
cyclic TS size
total yield (%) of crude products isolated
react
LP
BA
LP
[M + H]+ found
BA
[M + H]+ found
5a
5b
5c
12
13
14
87
93
92
4.72
5.00
3.03
95.29
85.21
96.97
0.00
9.79
0.00
6a
6b
6c
444.2
458.1
472.2
7a
7b
7c
−
663.2
−
a
Semiquantitative determination HPLC−MS. The area of ion-peak resulting from the sum of the intensities of the [M + H]+ and [M + Na]+ ions for
b
each compound was integrated (corrected for starting material). React = Reactant; LP = ligated peptide; BA = bis-acylated product.
98%), which were used both directly for ligation studies and also
Study of the Feasibility of O → N Acyl Migrations via a
as intermediates to prepare the monoisotetrapeptides 8a−e.
12-, 13- and 14-Membered Cyclic Transition States.
B
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Scheme 3. Synthesis of O-Acyl Isotetrapeptides 9a−e
Scheme 4. Acyl Migration of O-Acyl Isotetrapeptides 9a−e
Attempts to ligate 5b (Scheme 2) under aqueous conditions,
(pH 7.3, 1 M buffer strength, μw 50 °C, 50 W, 3 h), failed to yield
the ligated product via a 13-membered transition state. However
when the reaction was carried out under microwave irradiation in
C
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Table 2. Chemical Ligation of O-Acyl Isotripeptides 9a−e
ab
,
relative area (%)
product characterization by HPLC−MS
ligated peptide (LP) bis-acylated product (BA)
react
cyclic TS size
total crude yield (%) of products isolated
react
LP
BA
LP
[M + H]+ found
BA
[M + H]+ found
9a
9b
9c
9d
9e
15
16
17
18
19
90
86
91
88
94
0.54
3.62
0.1
94.6
87.0
96.8
99.9
98.1
4.88
9.43
3.10
0.00
0.00
10a
10b
10c
10d
10e
501.0
515.1
529.1
543.1
557.2
11a
11b
11c
11d
11e
706.0
720.1
734.2
−
0.1
1.91
−
a
Semiquantitative determination HPLC−MS. The area of ion-peak resulting from the sum of the intensities of the [M + H]+ and [M + Na]+ ions for
each compound was integrated (corrected for starting material). React = Reactant, LP = ligated peptide, BA = bis-acylated product
b
Scheme 5. Competitive Ligation Experiment
piperidine−DMF at 50 °C, 50 W for 1 h (Scheme 2), HPLC−
MS indicated the formation of 85% of the desired intramolecular
ligated products 6b (retention time 28.09 min), via the 13-
membered transition state together with bis-acylated product 7b
(10%, retention time 60.19 min) and 5% of the starting material
5b (retention time 37.10 min). The retention times and
fragmentation patterns of 5b and 6b were also studied in control
experiments (HPLC−MS of pure 5b). Thus HPLC−MS, via
(−)ESI-MS/MS, confirmed that compounds 5b and 6b, each
with MW 457, have very different fragmentation patterns,
proving the formation of the intramolecular ligated product 6b.
Similar O → N acyl migration via 12- and 14-membered cyclic
transition states were studied by irradiating compound 5a and 5c
in piperidine−DMF at 50 °C, 50 W for 3 h. The HPLC−MS
results are summarized in Table 1. The results indicate the
formation of compounds 6a−c and demonstrate that O- to N-
acyl group migrations via 12- and 14-membered transition states
are even more highly preferred compared to intermolecular
acylation then for the 13-membered cyclic transition state.
Preparation of the O-Acyl Isotetrapeptides 9a−e. Boc-
protected monoisotetrapeptides 8a−e were synthesized in
solution phase, by coupling the benzotriazolides of Boc-
protected Gly, β-Ala and GABA 1a−c with the unprotected
monoisotripeptides 5a−c at −10 °C in 96, 98 and 97% yields,
respectively. The Boc-group of 8a−e were removed by stirring
each with concentrated HCl in 1,4-dioxane for 2 h to afford the
HCl salts of the unprotected monoisotetrapeptides 9a−e.
mixtures. HPLC−MS, via (−)ESI-MS/MS, confirmed that the
ligated products 10a−e each produced different MS fragmenta-
tion patterns from those of the starting monoisohexapeptides
9a−e. The relative abundances of the crude ligated mixtures as
analyzed by analytical HPLC are shown in Table 2.
Competitive Ligation Experiment. To further support the
intramolecular nature of the ligation of N-terminus unprotected
O-isopeptides 5a−c and 9a−e to form native peptide 6a−c and
10a−e, respectively, by chemical ligation via a 12- to 19-
membered transition state, we carried out the chemical ligation
of N-terminus unprotected O-isotetrapeptide 9c in the presence
of 5 equiv of dipeptide 12 (H-Leu-Gly-OH) under aqueous
conditions (Scheme 5). HPLC−MS analysis of the isolated crude
product (S49, Supporting Information) confirmed the formation
of 96% of the desired ligation product 10c having a retention
time at 42.73 along with 3% of intermolecular bis-acylated
product 11c having a retention time at 56.75. The starting
material 9c (1%, retention time 40.20) was revealed as well. Z-
Protected tripeptide 13, which is the N-acylated product of
dipeptide 12, was not observed in the HPLC−MS analysis.
CONCLUSION
■
Efficient and convenient syntheses of novel O-acyl isopeptides
containing Tyr-residues and subsequent chemical ligation studies
have demonstrated that successful intramolecular O- to N-acyl
transfer via 12- to 14-membered transition states occurs in DMF-
Pip solution as well as 15- to 19-membered transition states in
aqueous solution. Microwave assisted isopeptide ligation offers
the following advantages: (i) short reaction times (3 h) at
moderate temperature (50 °C), (ii) traceless chemical ligation
from Tyr-containing isopeptides, and (iii) avoidance of ligation
auxiliaries. Compared with the classical native chemical ligation
approach, our methodology allows the isolation of the O-acyl
isopeptide intermediates, which may be useful in synthetic and
biological applications.
1
Compounds 8a−e and 9a−e were fully characterized by H,
13C NMR analysis (Scheme 3).
Study of the Feasibility of O → N Acyl Migrations via a
15- to 19-Membered Cyclic Transition States. Compounds
9a−e reacted under aqueous conditions (pH 7.3, 1 M buffer
strength, MW 50 °C, 50 W, 3 h) to produce the expected ligation
products 10a−e, in yields of 87−100% (Scheme 4). In the case of
9a, 9b and 9c the intermolecular bis-acylated products 11a, 11b
and 11c were also formed as shown by HPLC−MS (ESI)
analysis of the mixtures after ligation. Small amounts of
unreacted 9a−e were sometimes also present in the ligation
D
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Calcd for C27H33N3O9: C, 59.66; H, 6.12; N, 7.73. Found: C, 59.45; H,
6.22; N, 7.72.
EXPERIMENTAL SECTION
■
All commercial materials were used without further purification. All
solvents were reagent grade or HPLC grade. Melting points were
determined on a capillary point apparatus equipped with a digital
thermometer and are uncorrected. 1H NMR and 13C NMR spectra were
recorded in CDCl3, DMSO-d6 or CD3OD using a 300 MHz
spectrometer (with TMS as an internal standard). Chemical shifts are
reported in parts per million relative to residual solvent CDCl3 (1H, 7.26
ppm; 13C, 77.16 ppm), DMSO-d6 (1H, 2.50 ppm; 13C, 39.52 ppm),
CD3OD (1H, 3.31 ppm; 13C, 49.00 ppm). All 13C NMR spectra were
recorded with complete proton decoupling. All microwave assisted
reactions were carried out with a single mode cavity Discover Microwave
Synthesizer (CEM Corporation, NC). The reaction mixtures were
transferred into a 10 mL glass pressure microwave tube equipped with a
magnetic stirrer bar. The tube was closed with a silicon septum and the
reaction mixture was subjected to microwave irradiation (Discover
mode; run time 60 s.; PowerMax-cooling mode). HPLC−MS analyses
were performed on reverse phase gradient Phenomenex Synergi Hydro-
RP (2.1 × 150 mm; 5 um) + guard column (2 × 4 mm) or
Thermoscientific Hypurity C8 (5um; 2.1 × 100 mm + guard column)
using 0.2% acetic acid in H2O/methanol as mobile phases; wavelength =
254 nm; and mass spectrometry was done with electrospray ionization
(ESI).
Boc-β-Ala-L-Tyr(Z-L-Ala)-OH (4b). 0.91 g, 82%: mp 124−125 °C;
1H NMR (300 MHz, CDCl3) δ 7.33 (s, 5H), 7.15 (d, J = 7.7 Hz, 2H),
6.97 (d, J = 7.4 Hz, 1H), 6.76 (br s, 1H), 5.59 (d, J = 7.1 Hz, 1H), 5.29 (s,
1H), 5.14 (d, J = 12.0 Hz, 1H), 5.09 (d, J = 15.2 Hz, 1H), 4.80 (q, J = 6.1
Hz, 1H), 4.58 (t, J = 6.9 Hz, 1H), 3.39−3.24 (m, 2H), 3.19 (dd, J = 13.8,
5.1 Hz, 1H), 3.01 (dd, J = 13.4, 6.2 Hz, 1H), 2.36 (s, 2H), 1.53 (d, J = 6.9
Hz, 3H), 1.42 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 173.7, 171.9,
156.6, 155.9, 149.4, 136.2, 134.4, 130.6, 128.7, 128.3, 128.2, 121.3, 80.0,
67.2, 53.4, 50.0, 37.0, 36.4, 28.5, 18.5. Anal. Calcd for C28H35N3O9: C,
60.31; H, 6.33; N, 7.54. Found: C, 60.27; H, 6.59; N, 7.47.
Boc-GABA-L-Tyr(Z-L-Ala)-OH (4c). 0.96 g, 84%: mp 134−136 °C;
1H NMR (300 MHz, CDCl3) δ 7.41−7.22 (m, 7H), 7.00 (d, J = 8.3 Hz,
2H), 5.11 (s, 2H), 4.68 (dd, J = 9.2, 4.9 Hz, 1H), 4.41 (q, J = 7.3 Hz, 1H),
3.22 (dd, J = 14.0, 4.9 Hz, 1H), 3.09−2.88 (m, 3H), 2.21−2.13 (m, 2H),
1.65 (p, J = 7.1 Hz, 2H), 1.52 (d, J = 7.3 Hz, 3H), 1.43 (s, 9H); 13C NMR
(75 MHz, CDCl3) δ 175.4, 174.5, 173.4, 158.4, 150.9, 138.1, 136.4,
131.3, 129.4, 128.9, 128.7, 122.4, 79.9, 67.6, 54.8, 51.2, 40.6, 37.7, 34.0,
28.8, 27.3, 17.4. Anal. Calcd for C29H37N3O9: C, 60.93; H, 6.52; N, 7.35.
Found: C, 60.57; H, 6.57; N, 7.15.
Procedure for the Preparation of Hydrogen Chlorides of
Unprotected Isotripeptides 5a−c. Boc-protected isotripeptides
(4a−c) (1.00 mmol) were dissolved in 4 N HCl in 1,4-dioxane (15
mL) at 20 °C and stirred for 2 h. The reaction mixture was evaporated,
and the residue was recrystallized from diethyl ether to give the
corresponding hydrogen chloride salts of unprotected isodipeptides
(5a−c).
Procedure for Preparation of Dipeptides 3a−c. L-Tyr-OH (2)
(0.91 g, 5 mmol) and Boc-AA-Bt (1a−c) (5 mmol) were dissolved in
mixture of DMF (5−8 mL) and DBU (1.52 g, 1.49 mL, 10 mmol) and
left to stir at room temperature for 4 h. The reaction mixture was diluted
with 2 N hydrochloric acid, and product was extracted with EtOAc (3 ×
30 mL). The combined organic layers were washed with 2 N HCl (3 ×
10 mL) and dried over sodium sulfate. Evaporation and recrystallization
from diethyl ether gave desired dipeptides (3a−c).
H-Gly-L-Tyr(Z-L-Ala)-OH Hydrochloride (5a). 0.46 g, 96%: mp
73−75 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.92 (d, J = 8.0 Hz, 1H),
8.23 (br s, 3H), 7.99 (d, J = 6.6 Hz, 1H), 7.38−7.24 (m, 7H), 6.99 (d, J =
7.9 Hz, 2H), 5.06 (s, 2H), 4.56−4.45 (m, 1H), 4.31 (p, J = 6.8 Hz, 1H),
3.62−3.41 (m, 2H), 3.11 (dd, J = 13.7, 3.9 Hz, 1H), 2.91 (dd, J = 13.3,
9.0 Hz, 1H), 1.41 (d, J = 7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ
172.3, 171.4, 166.0, 156.1, 149.2, 137.0, 135.0, 130.4, 128.5, 128.0, 127.9,
121.3, 65.7, 53.9, 49.7, 42.8, 36.1, 16.8; HRMS (+ESI-TOF) m/z for
C22H26N3O7 [M − HCl + H]+ calcd 444.1765, found 444.1764.
H-β-Ala-L-Tyr(Z-L-Ala)-OH Hydrochloride (5b). 0.48 g, 98%: mp
1
Boc-Gly-L-Tyr-OH (3a). 1.42 g, 84%: mp 135−137 °C; H NMR
(300 MHz, CD3OD) δ 7.02 (d, J = 8.5 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H),
4.63 (dd, J = 7.3, 5.3 Hz, 1H), 3.73 (d, J = 17.1 Hz, 1H), 3.66 (d, J = 16.7
Hz, 1H), 3.08 (dd, J = 14.0, 5.2 Hz, 1H), 2.94 (dd, J = 14.0, 7.4 Hz, 1H),
1.43 (s, 9H); 13C NMR (75 MHz, CD3OD) δ 174.4, 172.1, 158.2, 157.3,
131.3, 128.5, 116.2, 80.8, 55.0, 44.4, 37.5, 28.7. Anal. Calcd for
C16H22N2O6: C, 56.80; H, 6.55; N, 8.28. Found: C, 56.77; H, 6.57; N,
8.30.
1
182−184 °C; H NMR (300 MHz, DMSO-d6) δ 8.55 (d, J = 8.0 Hz,
1H), 8.05−7.80 (m, 4H), 7.35−7.25 (m, 7H), 6.96 (d, J = 8.1 Hz, 2H),
5.05 (s, 2H), 4.47−4.38 (m, 1H), 4.30 (p, J = 6.5 Hz, 1H), 3.06 (dd, J =
13.8, 4.6 Hz, 1H), 2.90−2.76 (m, 3H), 2.65−2.35 (m, 3H), 1.40 (d, J =
7.2 Hz, 3H); 13C NMR (300 MHz, DMSO-d6) δ 172.8, 171.8, 169.5,
156.0, 149.0, 136.9, 135.4, 130.2, 128.4, 127.9, 127.8, 121.2, 65.6, 53.6,
49.7, 36.1, 35.2, 31.9, 16.8. Anal. Calcd for C23H28ClN3O7: C, 55.93; H,
5.71; N, 8.51. Found: C, 55.62; H, 5.82; N, 8.42.
Boc-β-Ala-L-Tyr-OH (3b). 1.39 g, 79%: mp 160−162 °C; 1H NMR
(300 MHz, CD3OD) δ 7.04 (d, J = 8.3 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H),
4.61 (dd, J = 8.7, 5.1 Hz, 1H), 3.23 (t, J = 6.7 Hz, 1H), 3.10 (dd, J = 14.0,
5.0 Hz, 1H), 2.86 (dd, J = 13.9, 8.8 Hz, 1H), 2.35 (t, J = 6.8 Hz, 2H), 1.42
(s, 9H); 13C NMR (75 MHz, CD3OD) δ 174.8, 173.6, 158.1, 157.2,
131.2, 128.9, 116.1, 80.2, 55.2, 37.9, 37.6, 36.9, 28.7. Anal. Calcd for
C17H24N2O6: C, 57.94; H, 6.86; N, 7.95. Found: C, 58.14; H, 7.05; N,
7.78.
H-GABA-L-Tyr(Z-L-Ala)-OH Hydrochloride (5c). 0.49 g, 97%: mp
164−166 °C; 1H NMR (300 MHz, CD3OD) δ 7.47−7.14 (m, 7H), 7.00
(d, J = 8.2 Hz, 2H), 5.12 (s, 2H), 4.72 (dd, J = 9.9, 4.4 Hz, 1H), 4.41 (q, J
= 7.2 Hz, 1H), 3.33−3.21 (m, 1H), 2.94 (dd, J = 13.9, 10.1 Hz, 1H),
2.88−2.65 (m, 2H), 2.42−2.20 (m, 2H), 1.82 (p, J = 7.0 Hz, 2H), 1.53
(d, J = 7.4 Hz, 3H); 13C NMR (300 MHz, CD3OD) δ 174.4, 174.3,
173.7, 158.5, 150.9, 138.1, 136.5, 131.4, 129.5, 129.0, 128.7, 122.5, 67.7,
54.8, 51.3, 40.2, 37.7, 33.4, 24.3, 17.3; HRMS (+ESI-TOF) m/z for
C24H30ClN3O7 [M − HCl + H]+ calcd 472.2078, found 472.2093.
Procedure for the Preparation of Isotetrapeptides 8a−c.
Unprotected isodipeptides (5a−c) (1 mmol) and Boc-AA-Bt (1 mmol)
were dissolved in DMF (5 mL) at −10 °C; TEA (0.15 g, 0.21 mL, 1.50
mmol) was added, and the reaction mixture was stirred at room
temperature for 8 h. The mixture was diluted with 2 N HCl and
extracted with EtOAc (3 × 20 mL). The combined organic layers were
washed with 2 N HCl (3 × 10 mL) and then dried over sodium sulfate.
Evaporation and recrystallization from diethyl ether gave isotetrapep-
tides (8a−e).
1
Boc-GABA-L-Tyr-OH (3c). 1.26 g, 69%: mp 65−67 °C; H NMR
(300 MHz, CD3OD) δ 7.04 (d, J = 8.5 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H),
4.60 (dd, J = 9.2, 5.0 Hz, 1H), 3.11 (dd, J = 13.9, 5.1 Hz, 1H), 2.98 (t, J =
6.8 Hz, 2H), 2.84 (dd, J = 14.0, 9.2 Hz, 1H), 2.23−2.14 (m, 2H), 1.66 (p,
J = 7.0 Hz, 2H), 1.43 (s, 9H); 13C NMR (75 MHz, CD3OD) δ 175.4,
174.9, 158.5, 157.2, 131.2, 129.1, 116.1, 79.9, 55.2, 40.6, 37.6, 34.0, 28.8,
27.3. Anal. Calcd for C18H26N2O6: C, 59.00; H, 7.15; N, 7.65. Found: C,
58.77; H, 7.59; N, 7.55.
Procedure for Preparation of Isotripeptides 4a−c. Boc-AA-L-
Tyr-OH (3a−c) (2 mmol) and Z-L-Ala-Bt (1d) (0.65 g, 2 mmol) were
dissolved in mixture of acetonitrile (30 mL) and TEA (0.30 g, 0.42 mL, 3
mmol) and left to stir at room temperature for 6 h. After evaporation the
reaction mixture was diluted with EtOAc (30 mL) and washed with 1 N
citric acid (3 × 30 mL). Organic layer was dried over sodium sulfate and
evaporated to give isotripeptides (4a−c).
1
Boc-Gly-L-Tyr(Z-L-Ala)-OH (4a). 0.77 g, 71%: mp 62−64 °C; H
Boc-Gly-Gly-L-Tyr(Z-L-Ala)-OH (8a). 0.37 g, 61%: mp 71−73 °C;
1H NMR (300 MHz, CD3OD) δ 7.49−7.12 (m, 7H), 7.00 (d, J = 7.4 Hz,
2H), 5.12 (s, 2H), 4.72−4.52 (m, 1H), 4.41 (q, J = 6.9 Hz, 1H), 3.98−
3.63 (m, 4H), 3.17 (dd, J = 13.8, 4.7 Hz, 1H), 3.03 (dd, J = 13.8, 9.1 Hz,
1H), 1.52 (d, J = 7.1 Hz, 3H), 1.45 (s, 9H); 13C NMR (75 MHz,
CD3OD) δ 174.5, 173.4, 172.9, 171.2, 158.5, 158.4, 150.9, 138.1, 136.3,
NMR (300 MHz, CDCl3) δ 7.33 (s, 5H), 7.15 (d, J = 8.1 Hz, 2H), 6.96
(d, J = 8.1 Hz, 2H), 5.62 (d, J = 7.8 Hz, 1H), 5.49 (s, 1H), 5.25−4.99 (m,
2H), 4.80 (s, 1H), 4.58 (p, J = 7.2 Hz, 1H), 3.92−3.56 (m, 2H), 3.27−
2.89 (m, 2H), 1.53 (d, J = 6.9 Hz, 3H), 1.41 (s, 9H); 13C NMR (75 MHz,
CDCl3) δ 173.6, 171.9, 170.1, 156.5, 156.0, 149.5, 136.2, 134.2, 130.6,
128.7, 128.2, 121.4, 80.6, 67.2, 53.8, 50.0, 36.9, 29.3, 28.4, 18.5. Anal.
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131.4, 129.4, 129.0, 128.7, 122.4, 80.8, 67.6, 55.3, 51.3, 44.8, 43.2, 37.7,
28.7, 17.4. Anal. Calcd for C29H36N4O10: C, 57.99; H, 6.04; N, 9.33.
Found: C, 57.72; H, 6.28; N, 9.32.
4.41 (q, J = 7.2 Hz, 1H), 3.39 (t, J = 6.7 Hz, 2H), 3.27−3.10 (m, 3H),
2.99 (dd, J = 14.2, 9.3 Hz, 1H), 2.56 (t, J = 6.4 Hz, 2H), 2.42 (hept, J =
7.8, 7.2 Hz, 3H), 1.52 (d, J = 7.3 Hz, 3H); 13C NMR (300 MHz,
CD3OD) δ 174.5, 173.6, 173.5, 172.1, 158.4, 150.9, 138.1, 136.3, 131.3,
129.4, 129.0, 128.7, 122.4, 67.6, 54.9, 51.3, 37.6, 37.2, 36.9, 36.2, 32.8,
17.4; HRMS (+ESI-TOF) m/z for C26H33ClN4O8 [M − HCl + H]+
calcd 529.2293, found 529.2301.
H-GABA-β-Ala-L-Tyr(Z-L-Ala)-OH Hydrochloride (9d). 0.52 g,
90%: mp 116−118 °C; 1H NMR (300 MHz, CD3OD) δ 7.38−7.23 (m,
7H), 7.01 (d, J = 8.2 Hz, 2H), 5.12 (s, 2H), 4.68 (dd, J = 9.1, 4.9 Hz, 1H),
4.41 (q, J = 7.2 Hz, 1H), 3.37 (t, J = 6.4 Hz, 2H), 3.22 (dd, J = 14.0, 4.7
Hz, 1H), 3.04−2.88 (m, 3H), 2.40 (q, J = 6.6 Hz, 2H), 2.30 (t, J = 6.9 Hz,
3H), 1.90 (p, J = 6.9 Hz, 3H), 1.52 (d, J = 7.3 Hz, 3H); 13C NMR (75
MHz, CD3OD) δ 174.4, 173.7, 173.5, 173.5, 157.3, 150.9, 138.1, 136.1,
131.1, 129.4, 128.9, 128.7, 122.5, 116.2, 67.5, 55.5, 50.9, 40.4, 37.6, 36.9,
36.1, 33.7, 24.3, 17.6; HRMS (+ESI-TOF) m/z for C27H35ClN4O8 [M −
HCl + H]+ calcd 541.2304, found 541.2315.
H-GABA−GABA-β-Ala-L-Tyr(Z-L-Ala)-OH (9e). 0.56 g, 95%: mp
81−83 °C; 1H NMR (300 MHz, CD3OD) δ 7.40−7.22 (m, 7H), 7.00
(d, J = 7.9 Hz, 2H), 5.12 (s, 2H), 4.68 (dd, J = 9.2, 4.8 Hz, 1H), 4.41 (q, J
= 7.2 Hz, 1H), 3.23 (dd, J = 14.0, 4.6 Hz, 1H), 3.11 (t, J = 6.6 Hz, 2H),
3.01−2.91 (m, 3H), 2.36 (t, J = 6.5 Hz, 2H), 2.21 (t, J = 7.2 Hz, 2H),
2.00−1.86 (m, 2H), 1.70 (p, J = 6.8 Hz, 2H), 1.52 (d, J = 7.2 Hz, 3H);
13C NMR (75 MHz, CD3OD) δ 175.3, 174.4, 173.6, 173.3, 158.3, 157.3,
150.9, 138.1, 136.1, 131.2, 131.1, 129.4, 128.9, 128.7, 122.4, 116.2, 67.6,
55.0, 51.3, 40.4, 39.7, 37.5, 33.9, 33.7, 26.5, 24.4, 17.4 HRMS (+ESI-
TOF) m/z for C28H37ClN4O8 [M − HCl + H]+ calcd 557.2606, found
557.2610.
General Procedure for Chemical Ligation of O-Acyl-
isotripeptides 5a−e in DMF−Piperidine. Isotripeptides (5a−c)
(0.20 mmol) were each dissolved in a mixture of DMF−piperidine (5
mL/1.5 mL), and the mixture was irradiated with microwave (50 °C, 50
W, 3 h) in a microwave tube. After cooling to room temperature the
reaction mixtures were acidified with 2 N HCl to pH = 1. Each mixture
was extracted with ethyl acetate (3 × 10 mL), the combined organic
extracts were dried over sodium sulfate, and the solvent was removed
under reduced pressure. Each ligation mixture was weighed, and then a
solution in methanol (1 mg mL−1) was analyzed by HPLC−MS.
General Procedure for Chemical Ligation of O-Acyl-isote-
trapeptides 9a−e in Buffer. Isotetrapeptides (9a−e) (0.20 mmol)
were each suspended in deoxygenated phosphate buffer (NaH2PO4/
Na2HPO4) (1 M, pH = 7.4, 8 mL) and irradiated with microwave (50
°C, 50 W, 3 h). Each reaction mixture was allowed to cool to room
temperature, acidified with 2 N HCl to pH = 1, and extracted with ethyl
acetate (3 × 10 mL). The combined organic extracts were dried over
Na2SO4, and the solvent was removed under reduced pressure. Each
ligation mixture was weighed, and a solution in methanol (1 mg mL−1)
was analyzed by HPLC−MS.
Boc-Gly-β-Ala-L-Tyr(Z-L-Ala)-OH (8b). 0.47 g, 76%: mp 108−110
°C; 1H NMR (300 MHz, CD3OD) δ 7.51−7.17 (m, 7H), 7.01 (d, J = 8.0
Hz, 2H), 5.11 (s, 2H), 4.69 (dd, J = 8.7, 5.0 Hz, 1H), 4.42 (q, J = 7.1 Hz,
1H), 3.65 (s, 2H), 3.38 (q, J = 6.2 Hz, 2H), 3.21 (dd, J = 13.8, 4.7 Hz,
1H), 2.97 (dd, J = 13.8, 9.1 Hz, 1H), 2.38 (q, J = 6.0 Hz, 2H), 1.51 (d, J =
7.2 Hz, 4H), 1.44 (s, 9H); 13C NMR (75 MHz, CD3OD) δ 174.4, 173.6,
173.4, 172.4, 158.3, 158.2, 150.9, 138.0, 136.3, 131.2, 129.4, 128.9, 128.7,
122.4, 80.7, 67.6, 54.8, 51.2, 44.6, 37.6, 36.8, 36.2, 28.7, 17.4. Anal. Calcd
for C30H38N4O10: C, 58.62; H, 6.23; N, 9.12. Found: C, 58.69; H, 5.77;
N, 9.36.
Boc-β-Ala-β-Ala-L-Tyr(Z-L-Ala)-OH (8c). 0.45 g, 71%: mp 125−
126 °C; 1H NMR (300 MHz, CD3OD) δ 7.50−7.15 (m, 7H), 7.00 (d, J
= 8.2 Hz, 2H), 5.11 (s, 2H), 4.70 (dd, J = 9.1, 4.9 Hz, 1H), 4.42 (q, J = 7.2
Hz, 1H), 3.43−3.15 (m, 5H), 2.97 (dd, J = 14.0, 9.3 Hz, 1H), 2.47−2.25
(m, 4H), 1.52 (d, J = 7.3 Hz, 3H), 1.42 (s, 9H); 13C NMR (75 MHz,
CD3OD) δ 174.5, 173.8, 173.6, 173.4, 158.3, 158.1, 150.9, 138.0, 136.3,
131.2, 129.4, 128.9, 128.7, 122.4, 80.1, 67.6, 54.8, 51.2, 38.0, 37.6, 37.3,
36.9, 36.3, 28.8, 17.4; HRMS (ESI) calcd for C31H40N4O10 [M + H]+
629.2817, found 629.2818.
Boc-GABA-β-Ala-L-Tyr(Z-L-Ala)-OH (8d). 0.40 g, 63%: mp 169−
171 °C; 1H NMR (300 MHz, CD3OD) δ 7.41−7.25 (m, 7H), 7.01 (d, J
= 8.4 Hz, 2H), 5.12 (s, 2H), 4.69 (dd, J = 9.2, 5.0 Hz, 1H), 4.42 (q, J = 7.3
Hz, 1H), 3.40−3.31 (m, 2H), 3.22 (dd, J = 13.9, 4.9 Hz, 1H), 3.12−2.90
(m, 3H), 2.48−2.31 (m, 2H), 2.14 (t, J = 7.5 Hz, 2H), 1.71 (p, J = 7.1 Hz,
2H), 1.52 (d, J = 7.3 Hz, 3H), 1.42 (s, 9H); 13C NMR (75 MHz,
CD3OD) δ 175.6, 174.5, 173.6, 173.4, 158.4, 151.0, 138.1, 136.4, 131.2,
129.4, 129.0, 128.7, 122.4, 79.9, 67.6, 54.9, 51.3, 40.8, 37.6, 36.9, 36.3,
34.3, 28.8, 27.2, 17.4. Anal. Calcd for C32H42N4O10: C, 59.80; H, 6.59; N,
8.72. Found: C, 59.83; H, 6.64; N, 8.69.
Boc-GABA−GABA-L-Tyr(Z-L-Ala)-OH (8e). 0.48 g, 73%: mp 98−
100 °C; 1H NMR (300 MHz, CD3OD) δ 7.45−7.15 (m, 7H), 7.00 (d, J
= 8.2 Hz, 2H), 5.11 (s, 2H), 4.70 (dd, J = 9.5, 4.9 Hz, 1H), 4.42 (q, J = 7.3
Hz, 1H), 3.23 (dd, J = 14.2, 4.9 Hz, 1H), 3.13−2.90 (m, 5H), 2.18 (t, J =
6.7 Hz, 4H), 1.70 (dp, J = 14.1, 6.8 Hz, 4H), 1.52 (d, J = 7.3 Hz, 3H),
1.43 (s, 9H); 13C NMR (75 MHz, CD3OD) δ 175.5, 175.3, 174.5, 173.4,
158.4, 150.9, 138.1, 136.3, 131.2, 129.4, 129.0, 128.7, 122.4, 79.9, 67.6,
54.7, 51.3, 40.8, 39.7, 37.7, 34.3, 34.1, 28.8, 27.3, 26.6, 17.4. Anal. Calcd
for C33H44N4O10: C, 60.35; H, 6.75; N, 8.53. Found: C, 60.08; H, 6.88;
N, 8.81.
Procedure for the Preparation of Hydrogen Chlorides of
Unprotected Isotetrapeptides 9a−e. Boc-protected isotetrapep-
tides (8a−c) (1 mmol) were dissolved in 4 N HCl in 1,4-dioxane (15
mL) and stirred at 0 °C for 2 h. After evaporation the residue was
recrystallized from diethyl ether to give the corresponding hydrogen
chloride salts of unprotected isodipeptides (9a−e).
H-Gly-Gly-L-Tyr(Z-L-Ala)-OH Hydrochloride (9a). 0.50 g, 93%:
1
ASSOCIATED CONTENT
mp 78−80 °C; H NMR (300 MHz, CD3OD) δ 7.41−7.19 (m, 7H),
■
7.00 (d, J = 7.8 Hz, 2H), 5.12 (s, 2H), 4.67 (dd, J = 7.5, 5.4 Hz, 1H), 4.41
(q, J = 7.3 Hz, 1H), 3.92 (d, J = 3.3 Hz, 2H), 3.72 (s, 3H), 3.20 (dd, J =
13.6, 4.2 Hz, 1H), 3.02 (dd, J = 13.6, 8.8 Hz, 1H), 1.52 (d, J = 7.2 Hz,
3H); 13C NMR (75 MHz, CD3OD) δ 174.3, 173.5, 170.8, 167.8, 158.5,
151.0, 138.1, 136.2, 131.4, 129.4, 129.0, 128.7, 122.4, 67.7, 55.2, 51.3,
43.0, 41.5, 37.7, 17.4; HRMS (+ESI-TOF) m/z for C24H29ClN4O8 [M −
HCl + H]+ calcd 501.1980, found 501.1984.
S
* Supporting Information
1H, 13C NMR and CHN/HRMS for all the novel compounds
and the chromatograms from the HPLC experiments. This
material is available free of charge via the Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: katritzky@chem.ufl.edu.
Notes
H-Gly-β-Ala-L-Tyr(Z-L-Ala)-OH Hydrochloride (9b). 0.46 g, 84%:
mp 104−106 °C; 1H NMR (300 MHz, CD3OD) δ 7.45−7.19 (m, 7H),
7.01 (d, J = 7.8 Hz, 2H), 5.11 (s, 2H), 4.69 (dd, J = 8.5, 4.6 Hz, 1H), 4.42
(q, J = 7.1 Hz, 1H), 3.65 (s, 2H), 3.44 (t, J = 6.3 Hz, 2H), 3.22 (dd, J =
13.6, 4.1 Hz, 1H), 2.99 (dd, J = 13.7, 9.3 Hz, 1H), 2.56−2.31 (m, 2H),
1.52 (d, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CD3OD) δ 174.6, 173.5,
173.4, 167.1, 158.4, 150.8, 138.0, 136.3, 131.3, 129.4, 128.9, 128.7, 122.4,
67.6, 54.9, 51.2, 41.6, 37.5, 36.9, 36.0, 17.4; HRMS (ESI) (+ESI-TOF)
m/z for C25H31ClN4O8 [M − HCl + H]+ calcd 513.1991, found
513.2004.
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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
H-β-Ala-β-Ala-L-Tyr(Z-L-Ala)-OH Hydrochloride (9c). 0.51 g,
91%: mp 114−116 °C; 1H NMR (300 MHz, CD3OD) δ 7.46−7.19 (m,
7H), 7.00 (d, J = 7.9 Hz, 2H), 5.11 (s, 2H), 4.68 (dd, J = 9.2, 5.0 Hz, 1H),
F
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technical and financial support of KAU. We also thank Abdulla
M. Asiri and Dr. C. D. Hall for helpful suggestions.
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