An alternate preparation of thioester resin linkers for solid-phase synthesis of peptide C-terminal thioacids

Article abstract of DOI:10.1016/S0040-4039(00)00266-5

An alternative preparation of thioester resin linkers for solid-phase synthesis of peptide C-terminal thioacids is presented. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00266-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2797–2800
An alternate preparation of thioester resin linkers for solid-phase
synthesis of peptide C-terminal thioacids
Alex S. Goldstein and Michael H. Gelb ∗
University of Washington, Departments of Chemistry and Biochemistry, Box 351700, Seattle, WA 98195-1700, USA
Received 12 January 2000; accepted 8 February 2000
Abstract
An alternative preparation of thioester resin linkers for solid-phase synthesis of peptide C-terminal thioacids is
presented. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: thioacids; thioesters; thiopeptides; supported reagents/reactions; peptide analogues/mimetics.
1. Introduction
The solid phase peptide synthesis of large peptides (greater than 60 amino acids) is problematic in
that it suffers from low yields and purification problems. To overcome these problems, researchers have
covalently joined unprotected, large peptide fragments using ‘native chemical ligation’ strategies. In one
of the first approaches developed, a globally deprotected C-terminal thioacid peptide is coupled to a
halogenated ‘N-terminal’ peptide in aqueous media.1,2 The newly created peptide contains a thioester
peptidomimetic bond that connects the fragments. The difficulty with this approach lies in the chemical
synthesis of the appropriate C-terminal thioacid-containing peptide. In a previously reported synthesis,
thioester (1) was prepared from 4-hydroxybenzophenone (2) via intermediate dibenzyl alcohol (3). This
alcohol was transformed to the corresponding chloride, which in turn could be converted to thioester (1)
by a one- or two-step route.3,4 The thioester (1) can be attached to an aminomethyl resin and elongated
using standard Boc-chemistry with the ultimate cleavage from the resin effected by treatment with HF
to give the C-terminal peptide thioacid. In our studies involving a novel peptide vaccine delivery system
based on lipid tubules, it became necessary to find a more reliable and efficient way to generate the
thioester (1).
∗
Corresponding author. Tel: 206 525 8405; fax: 206 616 4209; e-mail: gelb@chem.washington.edu (M. H. Gelb)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00266-5
tetl 16546

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2. Discussion
Repeated attempts to prepare the substituted dibenzyl alcohol (3) using the reported synthetic pathway
were unsuccessful. The substituted benzophenone derivative would not reduce under the given condi-
tions; therefore, an alternative pathway was devised (Fig. 1). 4-Hydroxybenzophenone (2) is initially
reduced to the corresponding alcohol by treatment with sodium borohydride in refluxing dioxane/water.
The phenol can then be selectively alkylated upon treatment with potassium carbonate and ethyl iodo-
acetate. The resulting ester is then converted to the corresponding carboxylic acid (3) by saponification.
Fig. 1.
The formation of the chloride, the next step in the reported thioester synthesis, also proved problematic.
We were unable to purify this very reactive intermediate. Instead, we generated the thioester linker (1)
directly by Lewis acid (zinc iodide) catalyzed coupling of the alcohol (3) with a thioacid.5 The required
aminothioacids were formed from the corresponding N-hydroxysuccinimidyl-esters (NHS-ester) either
by treatment with anhydrous H2S in triethylamine/dioxane4 (method A) or with NaHS in methanol
(method B). Both the aminothioacid and thioester linker formation conditions are quite mild. The
synthesis is amenable to a wide variety of chemical functionalities (Table 1) and should be compatible
with all protected amino acids commonly used in standard BOC chemistry. The optical rotations of the
thioester linkers (1) made directly from the alcohol (3) are identical to those made by coupling the NHS-
ester to a thiol linker.3
Table 1

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3. Experimental
1H NMR spectra were obtained in CDCl3 using a Bruker 300 MHz NMR spectrometer with tetra-
methylsilane as an internal standard. Silica gel (EM Science Silica Gel 60, 230–400 Mesh) was used for
all flash chromatography. TLC was performed using plates coated with 250 µm Silica Gel 60 F254 (EM
Science). All reagents were used as received.
3.1. Formation of thioacids6
Method A: A solution of triethylamine (3 equiv.) in p-dioxane at 0°C was saturated with anhydrous H2S
(gentle bubbling for ∼25 min). The NHS-ester (1 equiv., 13 mg/mL) was added and the solution stirred
for 40 min while warming to room temperature. The yellow solution was partially neutralized with 1N
HCl. By bubbling N2 through the solution for 20 min, excess H2S was removed and the mixture lightened
in color. The mixture was concentrated to quarter the volume and then acidified to pH=3 with 1N HCl.
The solution was diluted with 20 mL H2O and then extracted twice with 15 mL portions of EtOAc. The
organic layers were combined and washed with 3×15 mL H2O and once with 15 mL saturated NaCl
(aq.). The solvent was removed and the residue purifed by flash chromatography to provide the thioacids
as clear oils.
Method B: To NHS-ester in anhydrous MeOH (1 equiv., 10 mg/mL) was added (NaHS)·(H2O)1.5 (1
equiv.). The solution was stirred for 3 h and then acidified to pH=4 with 1N HCl. Water (20 mL) was
added and the solution was extracted with 30 mL EtOAc. The organic layers were combined and washed
with 10 mL H2O and 10 mL saturated NaCl. The solvent was removed and the residue purifed by flash
chromatography to provide the thioacids as clear oils.
3.2. Preparation of 4-benzoylphenoxyacetic acid (3)
To 4-hydroxybenzophenone (1.0 g, 5.0 mmol) in 100 mL 19:1 p-dioxane:H2O was added sodium
borohydride (0.4 g, 10.1 mmol). The solution was maintained at reflux for 14 h and then concentrated
to half the volume. The solution was acidified with 1 M H2SO4 until pH=3 and an additional 100 mL
H2O was added. The solution was extracted with 3×50 mL EtOAc and the combined organics were
washed with 50 mL saturated NaCl (aq.). The solvent was removed by rotary evaporation and the residue
partially purified by flash chromatography (25:1–8:1, CHCl3:MeOH) and those fractions containing the
reduced material were dried and then recrystallized from acetone/H2O to provide 4-benzoylphenol as
white needles (0.6 g, 60%): mp 160–162°C; Rf (9:1, CHCl3:MeOH) 0.48; 1H NMR 7.36–7.28 (m, 7H),
6.87 (d, 2H, ArOH, J=8.8 Hz), 5.80 (s, 1H, ArCH), 4.79 (s, 1H, ArOH), 2.19 (s, 1H, OH).
3.3. 4-Benzyloxyphenoxyacetic acid ethyl ester
To 4-benzoylphenol (0.6 g, 3.0 mmol) in 35 mL anhyd. DMSO was added anhydrous potassium
carbonate (0.8 g, 6.0 mmol) and ethyl iodoacetate (354 µL, 3.3 mmol). After stirring overnight, the
slurry was acidified to pH=4 with 1 M H2SO4. Water (25 mL) was added and the solution extracted
with 3×20 mL EtOAc. The organic layers were combined and washed with 25 mL 1 M H2SO4 and
25 mL H2O. The solvent was removed under reduced pressure and the residue was purified by flash
chromatography (20:1–9:1, CHCl3:MeOH) to provide the desired material as a clear oil (0.6 g, 68%): Rf
1
(9:1, CHCl3:MeOH) 0.58; H NMR 7.28 (m, 7H), 6.87 (d, 2H, J=8.8 Hz), 5.80 (s, 1H), 4.60 (s, 2H),
4.23 (q, 2H, J=7.3 Hz), 2.19 (s, 1H, OH), 1.29 (t, 3H, J=7.3 Hz).

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3.4. 4-Benzoylphenoxyacetic acid (3)
To 4-(α-hydroxybenzyl)phenoxyacetic ethyl ester (0.6 g, 2.1 mmol) in 100 mL MeOH and 5 mL H2O
was added sodium hydroxide (0.2 g, 5 mmol). After stirring overnight the mixture was acidified to pH=4
with 1 M H2SO4. Approximately half of the solvent was removed by rotary evaporation whereupon
an additional 75 mL H2O was added. The mixture was extracted with 3×50 mL EtOAc. The organic
layers were combined and washed with 40 mL saturated NaCl (aq). The solvent was removed under
reduced pressure and the residue purified by flash chromatography (20:1–9:1, CHCl3:MeOH) to provide
the desired material as a clear film (0.5 g, 100%): Rf (9:1:1 CHCl3:MeOH:HOAc) 0.56; 1H NMR 7.25
(m, 7H), 6.80 (d, 2H, J=8.8 Hz), 5.80 (s, 1H), 4.60 (s, 2H).
3.5. Formation of thioester linker (1)
To thioacid (1.1 equiv., 30 µmol/mL) and alcohol 3 (1.0 equiv.) in dry CH2Cl2 was added zinc iodide
(0.5 equiv.). The heterogeneous mixture was refluxed in an inert atmosphere for at least 17 h during which
it turned a light pink to red color. After cooling to room temperature the solution was filtered, and the
solvent removed under reduced pressure. The residue was purified by flash chromatography (30:1–8:1,
CHCl3:MeOH) to provide the thioester as oils.7
References
1. Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. USA 1981, 78, 4055–4058.
2. Schnolzer, M.; Kent, S. B. H. Science 1992, 256, 221–225
3. Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett. 1995, 38, 1217–1220.
4. Yamashiro, D.; Li, C. H. Int. J. Peptide Protein Res. 1988, 31, 322–334.
5. Gauthier, J. Y.; Bourdon, F.; Young, R. N. Tetrahedron Lett. 1986, 27, 15–18.
6. All aminothioacids have an identical Rf and 1H NMR to their oxyacid analogues.
7. All thioester linkers (1) have an Rf of 0.56 in 9:1:1 CHCl3:MeOH:HOAc. Their 1H NMR are identical to the superimposition
of the N-BOC-aminothioacid with 4-benzoylphenoxyacetic acid (3).