THAL, a sterically unhindered linker for the solid-phase synthesis of acid-sensitive protected peptide acids

Article abstract of DOI:10.1021/jo800535m

(Chemical Equation Presented) The 5-(4-hydroxyphenyl)-3,4- ethylenedioxythienyl alcohol (THAL, Thiophene Acid Labile) is described as a new linker for the solid-phase synthesis of peptide carboxylic acids. It is based on the electron-rich 3,4-ethylenedioxythenyl (EDOTn) moiety and allows the obtention of free and tert-butyl-protected peptides by cleavage with 90% and 0.5% TFA, respectively. This very high acid lability makes it useful for the synthesis of sensitive peptides. Free and tert-butyl- protected Leu-enkephalins have been synthesized as models to demonstrate the utility of the linker.

Full text of DOI:10.1021/jo800535m

THAL, a Sterically Unhindered Linker for the
Solid-Phase Synthesis of Acid-Sensitive Protected
Peptide Acids
Albert Isidro-Llobet,†,‡ Ulrik Boas,§ Knud J. Jensen,§
Mercedes Alvarez,* and Fernando Albericio*,†,‡,
,†,|
´
FIGURE 1. THAL-resin.
Institute for Research in Biomedicine, Barcelona Science
Park, UniVersity of Barcelona, Josep Samitier 1,
biologically interesting sensitive or complex peptides requires
the use of mild reagents for deprotection and cleavage opera-
tions. In this aspect, super acid-labile resins are of vital
importance because they allow the cleavage of very acid-
sensitive peptidic moieties or tert-butyl-protected peptide frag-
ments, which will be further modified after the cleavage. The
2-chlorotrityl chloride resin (2-CTC)6 has been the resin of
choice for most of these cases, and although it has given very
good results in a large number of complicated synthesis,7,8 it
has the drawback of its steric hindrance due to the bulkiness of
the 2-chlorotrityl moiety.
08028-Barcelona, Spain, CIBER-BBN, Networking Centre on
Bioengineering, Biomaterials and Nanomedicine, Barcelona
Science Park, Josep Samitier 1, 08028-Barcelona, Spain,
Department of Natural Sciences, Faculty of Life Sciences,
UniVersity of Copenhagen, Copenhagen, Denmark,
Laboratory of Organic Chemistry, Faculty of Pharmacy,
UniVersity of Barcelona, 08028-Barcelona, Spain, and
Department of Organic Chemistry, UniVersity of Barcelona,
Mart´ı i Franque´s 1, 08028-Barcelona, Spain
In previous work we demonstrated the utility of EDOTn
derivatives as super acid-labile protecting groups.9-11 In the
present work we propose 5-(4-hydroxyphenyl)-3,4-ethylene-
dioxythenyl alcohol (THAL) (Figure 1) as a sterically nonhin-
dered super acid-labile linker for the solid-phase synthesis of
peptide carboxylic acids, in particular as the C-terminal acid.
albericio@irbbarcelona.org;
mercedes.alVarez@irbbarcelon.org
ReceiVed March 8, 2008
5-(4-Hydroxyphenyl)-3,4-ethylenedioxythiophene-2-carbalde-
hyde (5) was prepared by formylation of 3,4-ethylenedioxy-
thiophene (2),9 followed by iodination and a Suzuki coupling
with p-hydroxyphenylboronic acid (Scheme 1), and it was
coupled to a conventional hydroxymethyl Merrifield polystyrene
resin. From the different coupling methods and resins tried, the
best approach was the coupling via formation of a trichloro-
acetimidate on the hydroxymethyl polystyrene resin. After the
coupling of the phenol, the resin was treated with DMF-H2O
to hydrolyze the remaining trichloroacetimidate. Then, it was
acetylated with Ac2O and DMAP, and the aldehyde was reduced
The 5-(4-hydroxyphenyl)-3,4-ethylenedioxythienyl alcohol
(THAL, Thiophene Acid Labile) is described as a new linker
for the solid-phase synthesis of peptide carboxylic acids. It
is based on the electron-rich 3,4-ethylenedioxythenyl (EDOTn)
moiety and allows the obtention of free and tert-butyl-
protected peptides by cleavage with 90% and 0.5% TFA,
respectively. This very high acid lability makes it useful for
the synthesis of sensitive peptides. Free and tert-butyl-
protected Leu-enkephalins have been synthesized as models
to demonstrate the utility of the linker.
(3) Fields, G. B.; Lauer-Fields, J. L.; Liu, R.-q.; Barany, G. In Synthetic
Peptides: A User’s Guide, 2nd ed.; Grant, G. A., Eds.; W. H. Freeman & Co.:
New York, NY, 2001; pp 93-219.
(4) Synthesis of Peptides and Peptidomimetics; Goodman, M., Felix, A.,
Moroder, L. A., Toniolo, C., Eds.; Houben-Weyl, Vol. E22a-e; Georg Thieme
Verlag: Stuttgart, Germany, 2002.
(5) The orthogonal concept is based on the use of independent classes of
protecting groups, removed by different mechanisms so that they may be removed
in any order and in the presence of all other types of groups. (a) Barany, G.;
Merrifield, R. B. J. Am. Chem. Soc. 1977, 99, 7363. (b) Barany, G.; Albericio,
F. J. Am. Chem. Soc. 1985, 107, 4936–4942.
(6) Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Yao, W.;
Schaefer, W. Tetrahedron Lett. 1989, 30, 3943–3946.
(7) Han, Y.-K.; Johnston, D. A.; Khatri, H. N. Synthesis of T-20 peptides.
PCT Int. Appl. (2006),WO 2006069727 A2 20060706, CAN 145:103960, AN
2006:653931.
At present most solid-phase peptide syntheses are performed
using the Fmoc/tBu orthogonal strategy.1-5 The obtention of
† Institute for Research in Biomedicine, University of Barcelona.
‡ CIBER-BBN.
§ University of Copenhagen.
| Faculty of Pharmacy, University of Barcelona.
(8) Gracia, C.; Isidro-Llobet, A.; Cruz, L. J.; Acosta, G. A.; Alvarez, M.;
Cuevas, C.; Giralt, E.; Albericio, F. J. Org. Chem. 2006, 71, 7196–7204.
Department of Organic Chemistry, University of Barcelona.
(1) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the
Synthesis of Peptides and Proteins; CRC: Boca Raton, FL, 1997. Fmoc Solid
Phase Peptide Synthesis; Chan, W. C., White, P. D., Eds.; Oxford University
Press: Oxford, U.K., 2000.
(2) Yokum, T. S.; Barany, G. In Solid Phase Synthesis. A Practical Guide;
Marcel Dekker Inc.: New York, NY, 2000; pp 79-102.
´
(9) Isidro-Llobet, A.; Just-Baringo, X.; Alvarez, M.; Albericio, F. Biopolymers
2008, 90, 444–449.
(10) Jessing, M.; Brandt, M.; Jensen, K. J.; Christensen, J. B.; Boas, U. J.
Org. Chem. 2006, 71, 6734–6741.
´
(11) Isidro-Llobet, A.; Alvarez, M.; Albericio, F. Tetrahedron Lett. 2008,
49, 3304–3307.
7342 J. Org. Chem. 2008, 73, 7342–7344
10.1021/jo800535m CCC: $40.75 2008 American Chemical Society
Published on Web 08/22/2008

SCHEME 1. Synthesis of 5-(4-Hydroxyphenyl)-3,4-ethylenedioxythiophene Carbaldehyde
SCHEME 2. Obtention of THAL-Resin
TABLE 1. Percentages of Cleavage of Fmoc-Gly-Phe-Leu-OH
from THAL-Resin
cleavage (%)
TFA-TES-CH2Cl2 (0.1:2:97.9), 10 min
TFA-TES-CH2Cl2 (0.5:2:97.5), 10 min
80
100
using NaBH4. (Scheme 2). Fmoc-Leu was coupled via formation
of the symmetric anhydride with DIC and DMAP. The loading
of the resin was 0.29 mmol/g (calculated by Fmoc UV titration
at λ ) 290 nm).
Acidolytic Release Studies. In order to test the acid lability
of the new linker, the tripeptide Fmoc-Gly-Phe-Leu-OH was
prepared on the resin. Aliquots of the peptide bonded to the
resin were treated with solutions containing 0.1% TFA, 0.5%
TFA, and 90% TFA.
It has been observed that phenyl-EDOTn derivatives are
completely removed using concentrations of 90% TFA.9 The
percentage of cleavage was calculated by HPLC analysis of the
crude cleavage mixtures using p-nitrobenzyloxycarbonyl-Ala-
OH (pNZ-Ala) as a standard. To determine the cleavage rates,
the same amount of a 1 mg/mL standard solution of pNZ-Ala
was added to the cleavage crude of the three aliquots. Then,
they were analyzed by HPLC, and the ratio of the areas from
the peaks corresponding to pNZ-Ala and Fmoc-Gly-Phe-Leu-
OH of the 0.1% and 0.5% TFA samples were compared with
the 90% of TFA sample to calculate the percentage of cleavage
in each case (Table 1).
FIGURE 2. HPLC (λ ) 220 nm) of 8 and 9. Linear gradients of
CH3CN (+0.036% TFA) into H2O (+0.045% TFA), from 30% to 100%
and from 0% to 80% in 15 min, respectively
The present THAL-resin allows release of peptide acids at
low concentrations of TFA (0.5% TFA within 10 min). Thus
tert-butyl-protected peptides and possibly very acid-sensitive
peptidic structures can easily be obtained. In addition, cleavage
with high concentrations of TFA provides unprotected peptides
with acceptable purities. As the new resin is based on a primary
alcohol, it is likely to cause little steric hindrance (Figure 3).
Synthesis of Protected and Unprotected Leu-Enkephalin:
H-Tyr(tBu)-Gly-Gly-Phe-Leu-OH (8) and H-Tyr-Gly-Gly-Phe-
Leu-OH (9) using THAL-Resin. The Leu-enkephalin was
chosen as a model peptide to demonstrate the utility of the new
derivatized resin. The coupling of Fmoc-Leu was performed as
indicated above. All remaining couplings were performed using
DIC and HOBt as coupling reagents. After the last Fmoc
removal, the peptide was cleaved from the resin using two
different conditions: 0.5% TFA for 10 min and 90% TFA for
1 h in order to obtain the tert-butyl-protected and free peptide,
respectively. In the former case the crude showed an excellent
purity by HPLC (Figure 2, left), whereas in the case of the latter
to obtain an acceptable purity washings with CH2Cl2 should be
performed (Figure 2, right). This lower purity at high concentra-
tions of TFA was also observed for the case of the 2-CTC resin
and suggests that the best method to obtain free peptides using
the THAL-resin is performing the cleavage of the protected
peptide with low concentrations of TFA and after filtering the
resin global deprotection by increasing the TFA concentration
to remove the tert-butyl-type protecting groups.
FIGURE 3. Comparison of the steric hindrance of (a) 2-chlorotrityl
chloride resin, (b) THAL-resin. The attachment of the peptide to the
resin is depicted in blue.
Experimental Section
(5-Iodo-3,4-ethylenedioxythiophene-2-carbaldehyde (4). Com-
pound 3 (1 g, 5.88 mmol) and N-iodosuccinimide (NIS) (1.59 g,
7.05 mmol) were dissolved in dry DMF (10 mL) and stirred at
120 °C until no starting material was detected by HPLC (usually
6-8 h). The reaction mixture was cooled to room temperature, Et2O
(100 mL) was added, and the resulting solution was washed with
H2O (3 × 100 mL). The organic portion was dried with MgSO4
and filtered, and the solution was stored at -20 °C and used within
24 h (dry product from this procedure was very unstable even at
low temperature).12
5-(4-Hydroxyphenyl)-3,4-ethylenedioxythiophene-2-carbalde-
hyde (5). DMF (50 mL) was added to a solution of 4 in diethyl
ether. The Et2O was evaporated, and more DMF was added to a
J. Org. Chem. Vol. 73, No. 18, 2008 7343

total volume of 150 mL. 4-Hydroxyphenylboronic acid (1.10 g,
7.94 mmol), Pd(PPh3)4 (416 mg, 0.360 mmol), and 10 mL of 2 M
aqueous Na2CO3 were added, and the mixture was stirred at 135
°C for 2 h. The course of the reaction was followed by TLC
(CH2Cl2). The reaction mixture was evaporated to dryness, an
aqueous solution of saturated NH4Cl (100 mL) was added, and the
mixture was extracted with AcOEt (3 × 125 mL). The organic
phase was dried with MgSO4, filtered, and evaporated to dryness.
MeOH (10 mL) was added to the crude obtained. The mixture was
filtered, and the light brown solid was dried under vacuum to yield
666 mg (43% total yield from 3) of the desired product: mp )
was added, and the suspension was stirred 15 min in the ice bath
and 24 h at room temperature. The yellow resin obtained was cooled
in an ice bath, and saturated aqueous NH4Cl was slowly added;
hydrogen evolution was observed. After 10 min, the NH4Cl was
removed, new NH4Cl was added, and the resin was left 5 min at 0
°C and 15 min more at room temperature. The resin was placed to
a 5-mL polypropylene syringe fitted with a polyethylene filter disk
and washed with H2O, MeOH, THF-H2O, THF, MeOH, and Et2O.
IR spectrometry was performed showing the absence of the
aldehyde absorption band.
General Solid-Phase Peptide Synthesis. Fmoc-Leu-OH (10
equiv) was coupled to 1 by forming the symmetric anhydride with
DIC (5 equiv) and DMAP (0.1 equiv) in CH2Cl2 (3 h).The loading
of the resin was determined by Fmoc UV titration (Fmoc group
was removed using piperidine-DMF (2:8) (1 × 1 min, 2 × 10
min). The remaining amino acids were coupled as Fmoc derivatives
(4 equiv), using DIC (4 equiv) and HOBt (4 equiv) as coupling
agents in DMF (1 h).
1
265.1-269.3 °C. H NMR (400 MHz, DMSO): δ 9.92 (s, 1H,
CHO), 9.81 (s, 1H, OH), 7.61 (d, 2H, 2CH arom, J ) 8.8 Hz),
6.83 (d, 2H, 2CH arom, J ) 8.8 Hz), 4.42 (m, 4H, CH2CH2). 13C
NMR (100 MHz, DMSO): δ 179.49 (CHO), 158.97 (C), 150.52
(C), 137.52 (C), 128.86 (CH), 122.87 (C), 116.58 (CH), 113.88
(C), 65.98 (CH2), 65.21 (CH2). HRMS (CI): m/z calcd for C13H9O4S
[M - H]- 261.0227, found 261.0225.
Resin Preparation. Trichloroacetimidate of the Hydroxy-
methyl Merrifield Resin (6). Hydroxymethylpolystyrene resin (300
mg, 0.98 mmol/g) was placed in a 5-mL polypropylene syringe
fitted with a polyethylene filter disk. The resin was then washed
with dry CH2Cl2 (5 × 0.5 min), swollen with CH2Cl2 (3 mL) for
20 min, and transferred to a tube equipped with a septum and under
nitrogen atmosphere. CCl3CN (427.4 µL, 14.5 equiv) was added,
and the tube was cooled in an ice bath. After 10 min of stirring
DBU (35.6 µL, 0.81 equiv) was added, and the resin was stirred
for 2 h at 0 °C. Then, the resin was transferred to the 5-mL syringe
and washed with CH2Cl2 (5 × 1 min) and Et2O (5 × 1min). An
aliquot of the resin was dried, and IR spectrometry was performed
showing the formation of the trichloroacetimidate. IR (KBr): ν )
H-Tyr(tBu)-Gly-Gly-Phe-Leu-OH (8) and H-Tyr-Gly-Gly-Phe-
Leu-OH (9) were obtained by treating the resin with TFA-TES-
CH2Cl2 (0.5:2:97.5) for 10 min and TFA-TES-CH2Cl2 (90:2:8) for
1 h, respectively. The cleavage solution for 8 was collected in H2O,
the TFA was evaporated, and after addition of CH3CN, the product
was characterized by HPLC (λ ) 220 nm), 95% purity. HRMS:
m/z calcd for C32H46N5O7 [M + Hl]+ 612.3397, found 612.3384.
In the case of the cleavage solution for 9, it was filtered, evaporated
to dryness, and dissolved in 0.1 N aqueous HCl (1 mL). This
aqueous phase was washed with CH2Cl2 (6 × 1 mL) to remove
apolar impurities and characterized by HPLC (λ ) 220 nm) and
LC-MS. The final product was obtained with 90% purity. HRMS:
m/z calcd for C28H38N5O7 [M + Hl]+ 556.2771, found 556.2762.
Cleavage Assays (Table 1). Aliquots of resin (10 mg) were taken
for cleavage assays. The first aliquot was treated with
TFA-TES-CH2Cl2 (90:2:8) (0.5 mL) for 1 h, the second aliquot
was treated with TFA-TES-CH2Cl2 (0.5:2:97.5) (1 mL) for 10
min, and the third with TFA-TES-CH2Cl2 (0.1:2:97.9) (0.5 mL)
for 10 min. The resulting cleavage solutions were separated from
the resin by filtration, evaporated to dryness, and dissolved in
H2O-CH3CN. Then, 0.8 mL of a 1 mg/mL standard solution of
pNZ-L-Ala-OH in CH3CN was added, and HPLC of the resulting
solution was performed.
3341, 1663 cm-1
.
5-(4-Hydroxyphenyl)-3,4-ethylenedioxythiophene-2-carbalde-
hyde Resin (7). The above obtained resin 6 was swelled in dry
CH2Cl2 for 20 min under nitrogen atmosphere, washed with dry
THF (3 × 1 min), and transferred to a tube equipped with a septum
and under nitrogen atmosphere. A suspension of 5 (154 mg, 2 equiv)
in dry THF was added, and the resin was stirred for 5 min. After
that BF3 ·Et2O (7.5 µL, 0.2 equiv) was added. The resin was gently
stirred for 4 h at room temperature in a shaker. The green resin
obtained was washed with THF, DMF, and CH2Cl2; treated with
DMF-H2O (2 × 15 min) in order to hydrolyze the remaining
trichloroacetimidate; and then washed with DMF (5 × 1 min), THF
anhydrous (5 × 1 min), and Et2O (5 × 1 min). An aliquot of the
resin was dried, and IR spectrometry was performed showing the
disappearance of the trichloroacetimidate and the formation of
Acknowledgment. This work was partially supported by
CICYT (CTQ2006-03794/BQU), Instituto de Salud Carlos III
(CB06_01_0074), the Generalitat de Catalunya (2005SGR
00662), the Institute for Research in Biomedicine, and the
Barcelona Science Park. A.I.-L. thanks the DURSI, Generalitat
de Catalunya and the European Social Funds for a predoctoral
fellowship.
the aldehyde. IR (KBr): ν ) 1649 cm-1
.
The resin was swollen in CH2Cl2 (15 min), washed with DMF
(5 × 30 s), and acetylated by treatment with Ac2O (554.8 µL, 20
equiv) and DMAP (2 equiv) in DMF (2 mL) for 45 min.
Supporting Information Available: 1H and 13C NMR
spectra of the prepared compounds and HPLC cleavage assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
5-(4-Hydroxyphenyl)-2-hydroxymethyl-3,4-ethylenedioxythio-
phene Resin, THAL-Resin (1). Resin 7 was swollen with THF for
15 min in a tube placed in an ice bath. NaBH4 (77.9 mg, 7 equiv)
(12) Alternatively, compound 2 can be prepared by a more laborious
protocol10 that provides a more stable product.
JO800535M
7344 J. Org. Chem. Vol. 73, No. 18, 2008