Phenyl-EDOTn derivatives as super acid labile carboxylic acid protecting groups for peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2008.03.074

A series of new 3,4-ethylenedioxy-2-thenyl (EDOTn) derived alcohols have been synthesized and evaluated as super acid labile carboxylic acid protecting groups. All the derivatives are labile to very low concentrations of TFA (0.01-0.5%).

Full text of DOI:10.1016/j.tetlet.2008.03.074

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3304–3307
Phenyl-EDOTn derivatives as super acid labile carboxylic
acid protecting groups for peptide synthesis
Albert Isidro-Llobet a,b, Mercedes Alvarez a,b,c, Fernando Albericio a,b,d,
´
*
a Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, Baldiri Reixac 10, 08028-Barcelona, Spain
b CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Josep Samitier 1, 08028-Barcelona, Spain
c Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028-Barcelona, Spain
d
´
Department of Organic Chemistry, University of Barcelona, Martı i Franque´s 1, 08028-Barcelona, Spain
Received 11 January 2008; revised 5 March 2008; accepted 14 March 2008
Available online 20 March 2008
Abstract
A series of new 3,4-ethylenedioxy-2-thenyl (EDOTn) derived alcohols have been synthesized and evaluated as super acid labile
carboxylic acid protecting groups. All the derivatives are labile to very low concentrations of TFA (0.01–0.5%).
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Peptide synthesis; EDOTn; EDOT; Acid labile protecting groups; Carboxylic acid protection
1. Introduction
remove the Fmoc group and, for most of the applications,
the conditions for its removal should leave tBu type groups
unaltered, allowing the obtention of tBu protected peptides
as well as other labile moieties present in the molecule such
as sugars.
3,4-Ethylenedioxythiophene (EDOT) is a highly elec-
tron-rich compound. Thus, protecting groups based on
3,4-ethylenedioxy-2-thenyl (EDOTn) should be very acid
labile due to the stabilization of the resulting thenyl carbo-
cation. EDOTn derivatives have recently been described as
very acid labile BAL-type linkers5 as well as amide back-
bone protectors,6 which can be removed by TFA–DCM
(1:99) and (95:5), respectively.
Nowadays, most peptide syntheses are carried out on
solid phase in the C to N direction with the a-carboxylate
of the C-terminal amino acid attached to the solid sup-
port.1 Nevertheless, the synthesis of several important pep-
tides involves side chain or backbone attachment to the
resin or synthesis in solution.2–4 In all these strategies, a
suitable protecting group for the C-terminal a-carboxylate
is mandatory. In addition to that, carboxylic acid protec-
tion is also necessary for the side chains of Asp and Glu
and broadly for other carboxylic groups.
In the case of solid phase peptide synthesis, the most
widely used strategy is the Fmoc/tBu strategy, which
involves a-amino temporary protection by the base labile
Fmoc group, side chain protection with trifluoroacetic acid
New EDOTn-derived compounds (1a–e) as very acid
labile carboxylic acid protecting groups are herein
described (Fig. 1).
t
(TFA) labile protecting groups, usually Bu type ones; and
cleavage from the resin also with TFA. Therefore, a suit-
able carboxyl protecting group for solid phase peptide syn-
thesis should be resistant to the base treatments used to
a: R= 3,4,5-trimethoxyphenyl
O
O
b: R= 3,4-dimethoxyphenyl
c: R=phenyl
d: R= H
HO
R
S
(1)
e: R= 4-methoxyphenyl
*
Corresponding author. Tel.: +34 93 403 70 88; fax: +34 93 403 71 26.
Fig. 1. EDOTn-derived alcohols prepared.
E-mail address: albericio@irbbarcelona.org (F. Albericio).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.03.074

A. Isidro-Llobet et al. / Tetrahedron Letters 49 (2008) 3304–3307
3305
In the previous works, the acid lability of EDOTn was
slightly increased by adding electron-donating substituents
via an inductive effect such as alkyl or alkylsulfide.5
In the present work, EDOTn has been functionalized
with electron-rich phenyl rings in order to increase the acid
lability by resonance effect. A series of esters of different
phenyl-EDOTn derivatives have been synthesized and their
lability to TFA evaluated, investigating also the effect of
adding electron-donating substituents to the phenyl ring.
From the two methods, A is the best because although
the iodination of 2 requires harsher conditions than the
iodination of 1d, the yields of the Suzuki reactions on com-
pound 3 are much higher than those on compound 6.8
Finally, for evaluation purposes alcohols 1a–e were
esterified with N-benzyloxycarbonylphenylalanine (Z–
Phe–OH) affording 5a–e esters in high yields.9
3. Removal assays10
2. Synthesis of alcohols based on electron-rich EDOTn
All the EDOTn derivatives prepared are labile to very
low concentrations of TFA (0.01–0.5% TFA in DCM)
(Table 1). Thus, they can be removed in the presence of
tBu type protecting groups. 1a–c and 1e are more acid
labile than 1d confirming that the conjugation provided
by the aromatic ring stabilizes the resulting carbocation.
Among the phenyl-EDOTn derivatives 1a–c and 1e, 1b is
more labile than 1e and the latter more labile than 1c.
Interestingly, 1a exhibits very similar lability to 1c. A
probable explanation is that the steric hindrance between
the three methoxy substituents makes them go out of the
aromatic ring plane and consequently their electron-donat-
ing effect decreases dramatically. Similar results have been
found in a protecting group for Arg.11 To corroborate this
hypothesis, UV spectra of compounds 4a–c were registered
Two different synthetic methods were tested. In Method
A (Scheme 1), the commercially available EDOT was
formylated in high yield using n-BuLi and DMF as
described in the previous works of the group6 yielding 2.
This latter was iodinated by reaction with N-iodosuccini-
mide (NIS) in DMF to afford 3. Suzuki–Miyaura reaction7
of the iodinated aldehydes with different phenylboronic
acids yielded furnished 4a–c, which were reduced with
NaBH4 to the corresponding alcohols 1a–c. In Method
B, compound 2 was reduced with NaBH4 to alcohol 1d.
Then, it was protected as acetate and iodinated with NIS
in DMF. The iodide was converted to 1e by reaction with
4-methoxyphenyl boronic acid (Scheme 2).
R1
R2
3R
OH
B
OH
O
O
O
O
O
O
O
NIS
i) nBuLi (1 eq)
(1.2 eq)
ii) DMF (1.9 eq)
Pd(PPh3)4, Na2CO3
H
H
DMF
THF
S
S
I
S
T=120 oC, 6 h
DMF-H2O (95:5)
T=130 oC, 2 h
O
O
3
2
O
R1
O
O
Z-Phe-OH (1.2 eq)
EDC (1.2 eq)
O
O
R1
R2
H
R1
R2
NaBH4
MeOH
HO
R2
S
DMAP (0.1 eq)
O
Z-Phe-
S
O
S
R3
Dry DCM
4a R1=R2=R3= OMe
4b R1=R2= OMe, R3= H
4c R1=R2=R3= H
R3
R3
5a-c
1a-c
Scheme 1. The synthesis of N-(benzyloxycarbonyl)phenylalanine (Z–Phe–OH) esters of EDOTn derivatives, Method A.
i) Ac2O, DMAP, rt, 30 min
ii) NIS (1.2 eq), DMF, rt, 3 h
O
O
O
O
O
O
NaBH4
MeOH
H
HO
O
AcO
S
2
S
1d
I
S
O
6
OH
OH
MeO
B
O
O
O
Z-Phe-OH (1.2 eq)
EDC (1.2 eq)
Pd(PPh3)4, Na2CO3
DMAP (0.1 eq)
Z-Phe-O
HO
S
S
OMe
DMF-H2O (95:5)
T=130 oC, 2 h
OMe
Dry DCM
5e
1e
Scheme 2. The synthesis of N-(benzyloxycarbonyl)phenylalanine (Z–Phe–OH) esters of EDOTn derivatives, Method B.

3306
A. Isidro-Llobet et al. / Tetrahedron Letters 49 (2008) 3304–3307
Table 1
3. Jensen, K. J.; Alsina, J.; Songster, M. F.; Vagner, J.; Albericio, F.;
Acid labilities of the different EDOTn derivatives
Barany, G. J. Am. Chem. Soc. 1998, 120, 5441–5452.
´
4. Jou, G.; Gonzalez, I.; Albericio, F.; Lloyd-Williams, P.; Giralt, E. J.
1a (%) 1b (%) 1e (%) 1c (%) 1d (%)
Org. Chem. 1997, 62, 354–366.
0.01% TFA, t = 10 min
0.01% TFA, t = 1 h
0.5% TFA, t = 5 min
0.5% TFA, t = 1 h
5
—
100
—
35
87
100
—
20
68
100
—
3
—
96
—
0
5. Jessing, M.; Brandt, M.; Jensen, K. J.; Cristensen, J. B.; Boas, U. J.
Org. Chem. 2006, 71, 6734–6741.
—
´
59
100
6. Isidro-Llobet, A.; Just-Baringo, X.; Alvarez, M.; Albericio, F.
Biopolymers. 2008, doi:10.1002/bip.20823.
7. Topics in Current Chemistry; Miyaura, N., Ed.; Springer Verlag:
Berlin Heidelbert, 2002.
8. Method A: 5-Iodo-3,4-ethylenedioxythiophene-2-carbaldehyde (3).
showing the following kmax: 362.0, 373.8 and 339.4 nm,
respectively. The decrease in kmax (hypsochromic shift) of
compound 4a compared to compound 4b is probably due
to the fact that in compound 4a the lone pairs of the meth-
oxy groups are less delocalized into the aromatic nucleus.
Compound
2 (510 mg, 3 mmol) and N-iodosuccinimide (NIS)
(810 mg, 3.6 mmol) were dissolved in dry DMF (5 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.
Diethylether (60 mL) was added and the resulting solution was
washed with H2O (3 Â 50 mL). The organic portion was dried over
MgSO4 and filtered; the solution was then stored at À20 °C and used
within 24 h maximum. (The dry product is very unstable even at low
temperatures). A small aliquot was characterized by 1H NMR
(400 MHz, CDCl3): d = 9.78 (s, 1H), 4.36 (s, 4H) HPLC.
5-(3,4,5-Trimethoxyphenyl)-3,4-ethylenedioxythiophene-2-carbaldehyde
(4a): DMF (25 mL) was added to the above obtained solution of 3 in
diethyl ether (60 mL). The diethyl ether was evaporated and more
DMF was added to reach a total volume of 85 mL, then 3,4,5-
trimethoxyphenylboronic acid (859 mg, 4.05 mmol), Pd(PPh3)4
(212 mg, 0.184 mmol), and 2 M aqueous Na2CO3 (5.4 mL) were also
added and the mixture was stirred at 135 °C for 4 h. The course of the
reaction was followed by TLC (EtOAc–hexane; 1:1). The reaction
mixture was evaporated to dryness, DCM (100 mL) was added and
the solution was washed with H2O (3 Â 100 mL). The organic phase
was dried over MgSO4 and evaporated to dryness. The crude obtained
was purified by silica gel chromatography (hexane, EtOAc) to render
620 mg of 4a (62% yield). Mp 146.3–149.8 °C. 1H NMR (400 MHz,
CDCl3): d = 9.93 (s, 1H), 7.02 (s, 2H), 4.42 (m, 4H), 3.9 (s, 6H), 3.88
(s, 3H).
5-(3,4-Dimethoxyphenyl)-3,4-ethylenedioxythiophene-2-carbaldehyde
(4b): 630 mg (69% yield, 90% purity). Mp 185.4–190.3 °C. 1H NMR
(400 MHz, CDCl3): d = 9.92 (s, 1H), 7.40 (dd, 1H, J = 8.4 and
2.1 Hz), 7.31 (d, 1H, J = 2.1 Hz), 6.90 (d, 1H, J = 8.4 Hz), 4.40 (m,
4H), 3.93 (s, 3H), 3.92 (s, 3H).
5-Phenyl-3,4-ethylenedioxythiophene-2-carbaldehyde (4c): 474.1 mg
(64% yield, 90% purity) mp 135.2–139.3 °C. 1H NMR (400 MHz,
CDCl3): d = 9.94 (s, 1H), 7.80 (dd, 2H, J = 7.2 and 1.4 Hz), 7.37 (m,
3H), 4.41 (m, 4H).
5-(3,4,5-Trimethoxyphenyl)-3,4-ethylenedioxythenyl alcohol (1a):
Compound 4a (150 mg, 0.45 mmol) was dissolved in MeOH (5 mL)
and the resulting suspension was cooled in an ice bath. NaBH4
(135.1 mg, 3.57 mmol) was added. The evolution of H2 was observed
and the initial suspension became a solution which was stirred at room
temperature for 1 h. After that, H2O (20 mL) was added and the pH
adjusted to 8 by adding NH4Cl; DCM extractions were then carried out
(3 Â 20 mL). The organic extracts were dried with MgSO4, evaporated
to dryness, and dried in the vacuum desiccator to get rid of all the
MeOH. 133.6 mg of a solid was obtained (89% yield, 90% purity).
1H NMR (400 MHz, DMSO): d = 6.86 (s, 2H), 5.29 (t, 1H,
J = 5.6 Hz), 4.46 (d, 2H, J = 5.6 Hz), 4.26 (m, 4H), 3.78 (s, 6H),
3.65 (s, 3H).
4. Orthogonality to the Fmoc group
Nowadays, most peptides synthesized on solid phase are
prepared using the Fmoc/tBu strategy. Therefore, it is
interesting to check how resistant alcohols 1a–e are used
to the piperidine-mediated removal of the Fmoc group.
3,4-Ethylenedioxythenyl
N-(benzyloxycarbonyl)phenyl-
alaninate (5d) was chosen as a model because it contains
the least electron-rich system and can, therefore, be the
most base labile. 5d was treated with piperidine-DMF
(2:8) for 48 h. HPLC analysis revealed that only 5% of
EDOTn removal took place, confirming that the EDOTn
derivatives described are compatible and orthogonal with
the Fmoc group.
5. Conclusions
In conclusion, the new PhEDOTn carboxyl protecting
groups can be removed using very low concentrations of
TFA, being rather stable to the standard protocols to
remove the Fmoc group. These groups can be useful alter-
natives for the preparation of complex peptides. Further-
more, the unexpected stability of 1c should be useful for
the design of new protecting groups.
Acknowledgments
We thank Professor Knud J. Jensen and Dr. Ulrik Boas
for fruitful discussions. 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 Biomedi-
cine, and the Barcelona Science Park. AI-L thanks the
DURSI, Generalitat de Catalunya and the European Social
Funds for a predoctoral fellowship.
5-(3,4-Dimethoxyphenyl)-3,4-ethylenedioxythenyl alcohol (1b): 129.7
mg (86% yield, 95% purity) 1H NMR (400 MHz, DMSO): d = 7.16
(d, 1H, J = 2.1 Hz), 7.13 (dd, 1H, J = 8.4 Hz, J = 2.1 Hz), 6.95 (d,
1H, J = 8.4 Hz), 5.25 (t, 1H, J = 5.6 Hz), 4.45 (d, 2H, J = 5.6 Hz),
4.25 (m, 4H), 3.76 (s, 3H), 3.75 (s, 3H).
References and notes
5-Phenyl-3,4-ethylenedioxythenyl alcohol (1c): 92.5 mg (84% yield,
85% purity) 1H NMR (400 MHz, DMSO): d = 7.61 (dd, 2H,
J = 8.4 Hz and 1.1 Hz), 7.36 (dd, 2H, J = 7.4 and 7.4 Hz), 7.20 (dd,
1H, J = 7.8 and 7.8 Hz), 5.30 (t, 1H, J = 5.6 Hz), 4.47 (d, 2H,
J = 5.6 Hz), 4.27 (m, 4H).
´
1. Tulla-Puche, J.; Bayo-Puxan, N.; Moreno, J. A.; Francesch, A. M.;
´
Cuevas, C.; Alvarez, M.; Albericio, F. J. Am. Chem. Soc. 2007, 129,
5322–5323.
´
2. Kates, S. A.; Sole, N. A.; Johnson, C. R.; Hudson, D.; Barany, G.;
Albericio, F. Tetrahedron Lett. 1993, 34, 1549–1552.

A. Isidro-Llobet et al. / Tetrahedron Letters 49 (2008) 3304–3307
3307
3,4-Ethylenedioxythenyl alcohol (1d): 2 g, (99% yield, 99% purity). 1H
NMR (400 MHz, CDCl3): d = 6.29 (s, 1H), 4.66 (s, 2H), 4.21 (m, 4H).
Method B: 5-Iodo-3,4-ethylenedioxythenyl acetate (6) 3,4-Ethylenedi-
oxythenyl acetate: 1d (700 mg, 4.07 mmol) was dissolved in Ac2O
(3 mL) and DMAP (49 mg, 0.41 mmol) was added. The reaction
mixture was stirred for 30 min at room temperature, poured into
saturated aqueous Na2CO3 (25 mL), and extracted with EtOAc
(20 mL). The organic layer was washed with saturated aqueous
Na2CO3 (6 Â 15 mL) and 0.1% HCl in H2O (3 Â 20 mL), dried with
MgSO4, and then evaporated to dryness to yield 760 mg of an oil
(87% yield).
DMAP (4.8 mg, 0.04 mmol) were dissolved in dry DCM (1.5 mL).
The reaction mixture was stirred for 90 min and checked by TLC
(DCM-MeOH, 95:5). After that, EtOAc (25 mL) was added, and the
organic layer was washed with saturated aqueous Na2CO3
(3 Â 25 mL) and H2O (6 Â 25 mL). The organic phase was dried
with MgSO4 and evaporated to dryness to yield 245.1 mg of an oil
(98% yield, 80% purity).
1H NMR (400 MHz, DMSO): d = 7.83 (d, 1H, J = 8.2 Hz), 7.25 (m,
10H), 6.89 (s, 2H), 5.09 (s, 2H), 4.96 (m, 3H), 4.30 (m, 4H), 3.78 (s,
6H), 3.66 (s, 3H), 3.0 (dd, 1H, J = 13 and 5.0 Hz) 2.86 (dd, 1H,
J = 13.9 Hz and 10.2 Hz).
5-(3,4-Dimethoxyphenyl)-3,4-ethylenedioxythenyl-N-(benzyloxycarb-
onyl)phenylalaninate (5b): 150.6 mg, (78% yield, 70% purity) 1H
NMR (400 MHz, DMSO): d = 7.82 (d, 1H, J = 8.1 Hz), 7.21 (m,
12H), 6.89 (d, 1H, J = 8.1 Hz), 5.09 (s, 2H), 4.96 (m, 3H), 4.29 (m,
4H), 3.76 (s, 3H, CH3), 3.75 (s, 3H), 3.01 (dd, 1H, J = 13.8 and
5.2 Hz) 2.85 (dd, 1H, J = 13.8 Hz and 10.1 Hz).
5-Phenyl-3,4-ethylenedioxythenyl-N-(benzyloxycarbonyl)phenylalanin-
ate (5c): 163.9 mg, (91% yield, 75% purity). 1H NMR (400 MHz,
DMSO): d = 7.83 (d, 1H, J = 8.2 Hz), 7.60 (m, 2H), 7.28 (m, 13H),
5.01 (m, 5 H), 4.29 (m, 4 H), 3.01 (dd, 1H, J = 13.8 Hz and 5.2 Hz),
2.85 (dd, 1H, J = 13.8 and 10.1 Hz).
1H NMR (400 MHz, CDCl3): d = 6.35 (s, 1H), 5.11 (s, 2H), 4.22 (m,
4H), 2.08 (s, 3H).
5-Iodo-3,4-ethylenedioxythenyl acetate (6) 3,4-Ethylenedioxythenyl
acetate (760 mg, 3.56 mmol) was dissolved in dry DMF(5.5 mL),
and NIS (958 mg, 4.27 mmol) was added. After 3 h of stirring at room
temperature, Et2O (75 mL) was added and the resulting solution was
washed with H2O (3 Â 100 mL). The organic layer was dried with
MgSO4, filtered and a small aliquot was taken for 1H NMR. DMF
(10 mL) was added to the remaining solution before removing Et2O
(the dry product is unstable) and this solution was immediately used
for the next reaction.
1H NMR (400 MHz, CDCl3): d = 5.07 (s, 2H), 4.25 (4H), 2.07 (s, 3H).
5-(4-Methoxyphenyl)-3,4-ethylenedioxythenyl alcohol (1e): DMF
(90 mL) was added to the above-mentioned DMF solution of 6.
4-methoxyphenylboronic acid (810 mg, 5.33 mmol), Pd(PPh3)4
(278 mg, 0.241 mmol), and 3.5 M aqueous Na2CO3 (8 mL) were
successively added and the mixture was stirred at 135 °C for 5 h. The
course of the reaction was followed by TLC (AcOEt–hexane, 1:1).
The reaction mixture was evaporated to dryness. Et2O (100 mL) was
added and the solution was washed with H2O (3 Â 100 mL). Et2O
(40 mL) was added to the organic phase and it was washed with brine
(3 Â 100 mL). Then it was dried with MgSO4 and evaporated to
dryness. The crude obtained was purified by column chromatography
(hexane, AcOEt) and 97 mg of an orange solid was obtained (10%
yield).
3,4-Ethylenedioxythenyl-N-(benzyloxycarbonyl)phenylalaninate (5d):
592 mg, (87% yield, 90% purity) 1H NMR (400 MHz, CDCl3): d
7.27 (m, 8H), 7.03 (m, 2H), 6.37 (s, 1H, CH), 5.12 (m, 3H), 4.68 (m,
1H), 4.19 (m, 4H), 3.11 (m, 2H).
5-(4-Methoxyphenyl)-3,4-ethylenedioxythenyl-N-(benzyloxycarbonyl)-
phenylalaninate (5e): 67.4 mg, (82% yield, 80% purity). 1H NMR
(400 MHz, CDCl3): d = 7.62 (d, 2H, J = 8.9 Hz), 7.33 (m, 6H), 7.19
(m, 3H), 7.05 (m, 2H), 6.91 (d, 2H, J = 8.9 Hz), 5.25–5.06 (m, 5H),
4.70 (m, 1H), 4.28 (m, 4H), 3.83 (s, 3H), 3.12 (m, 2H).
10. For each removal test, 1 mg of amino acid was treated with a solution
of the corresponding concentration of TFA with 2% of triethylsilane
as a scavenger. In all the solutions, a minimum of 2 equiv of TFA was
used. The reactions were stopped by adding 2 equiv of pyridine per
equivalent of TFA, evaporated, dissolved in H2O–AcCN, and
analyzed by HPLC. The removal percentage was calculated by
comparing the areas at k = 220 nm of the peaks corresponding to the
free and protected amino acid.
1H NMR (400 MHz, CDCl3): d = 7.61 (d, 2H, J = 8.9 Hz), 6.90 (d,
2H, J = 8.9 Hz), 4.37 (d, 2H, J = 4.4 Hz), 4.27 (m, 4H), 3.82 (s, 3H).
9. 5-(3,4,5-Trimethoxyphenyl)-3,4-ethylenedioxythenyl-N-(benzyloxy-
carbonyl)phenylalaninate (5a): Compound 1a (130 mg, 0.39 mmol),
Z–Phe–OH (140 mg, 0.47 mmol), EDC (145 mg, 0.47 mmol), and
11. Fujino, M.; Wakimasu, M.; Kitada, C. Chem. Pharmacol. Bull. 1981,
29, 2825–2831.