Thiophene backbone amide linkers, a new class of easily prepared and highly acid-labile linkers for solid-phase synthesis

Article abstract of DOI:10.1021/jo060687r

Solid-phase synthesis is of tremendous importance for small-molecule and biopolymer synthesis. Linkers (handles) that release amide-containing products after completion of solid-phase synthesis are widely used. Here we present a new class of highly acid-labile backbone amide linkers (BAL handles) based on 3,4-ethylenedioxythiophene (EDOT), which we have termed T-BAL. These thiophene linkers are synthesized in three convenient steps from commercially available EDOT. In the linker design, the spacer was introduced to the EDOT core either via a carbon-carbon bond or via a thioether linkage. Introduction of the spacer via a C-C bond was performed by a chemoselective Negishi coupling without transient protection of the aldehyde group to provide the T-BAL1 handle. Introduction via a thioether linkage was performed by a facile nucleophilic aromatic substitution between the brominated EDOT aldehyde and unprotected mercapto acids to provide T-BAL2 and T-BAL3 handles. The minimal use of protecting groups gave the corresponding linker molecules in few synthetic steps and in good yields. After anchoring of the linker to a polymeric support, introduction of the first amino acid was achieved by reductive amination, giving a secondary amine. A following acylation of the secondary amine with a symmetrical amino acid anhydride resulted in a backbone amide linkage between the handle and the growing substrate (e.g., peptide chain). After solid-phase synthesis, the substrates could be released from the resin by either low acid conditions using 1% TFA in CH₂Cl₂ or high acid conditions such as 50% TFA in CH₂Cl₂. Peptide thioesters could be released from the T-BAL1 handle under very mild conditions using aqueous acetic acid. Tert-butyl based protecting groups, tert-butyl esters, tert-butyl ethers, and Boc groups, as well as dimethyl acetals were relatively stable to these mild conditions for release of the peptides.

Full text of DOI:10.1021/jo060687r

Thiophene Backbone Amide Linkers, a New Class of Easily
Prepared and Highly Acid-Labile Linkers for Solid-Phase Synthesis†
Mikkel Jessing,‡,⊥ Malene Brandt,§,⊥ Knud J. Jensen,* Jørn B. Christensen, and
,§
‡
,
‡,§,|
Ulrik Boas*
Department of Chemistry, UniVersity of Copenhagen, UniVersitetsparken 5, DK-2100, Denmark,
Department of Natural Sciences, Royal Veterinary and Agricultural UniVersity, ThorValdsensVej 40,
DK-1871, Denmark, and Department of Veterinary Diagnostics and Research, Danish Institute for Food
and Veterinary Research (DFVF), B u¨ lowsVej 27, DK-1790, Denmark
kjj@kVl.dk; ubo@dfVf.dk
ReceiVed March 30, 2006
Solid-phase synthesis is of tremendous importance for small-molecule and biopolymer synthesis. Linkers
(
handles) that release amide-containing products after completion of solid-phase synthesis are widely
used. Here we present a new class of highly acid-labile backbone amide linkers (BAL handles) based on
,4-ethylenedioxythiophene (EDOT), which we have termed T-BAL. These thiophene linkers are
3
synthesized in three convenient steps from commercially available EDOT. In the linker design, the spacer
was introduced to the EDOT core either via a carbon-carbon bond or via a thioether linkage. Introduction
of the spacer via a C-C bond was performed by a chemoselective Negishi coupling without transient
protection of the aldehyde group to provide the T-BAL1 handle. Introduction via a thioether linkage was
performed by a facile nucleophilic aromatic substitution between the brominated EDOT aldehyde and
unprotected mercapto acids to provide T-BAL2 and T-BAL3 handles. The minimal use of protecting
groups gave the corresponding linker molecules in few synthetic steps and in good yields. After anchoring
of the linker to a polymeric support, introduction of the first amino acid was achieved by reductive
amination, giving a secondary amine. A following acylation of the secondary amine with a symmetrical
amino acid anhydride resulted in a backbone amide linkage between the handle and the growing substrate
(e.g., peptide chain). After solid-phase synthesis, the substrates could be released from the resin by either
low acid conditions using 1% TFA in CH Cl or high acid conditions such as 50% TFA in CH Cl
2
2
2
2
.
Peptide thioesters could be released from the T-BAL1 handle under very mild conditions using aqueous
acetic acid. Tert-butyl based protecting groups, tert-butyl esters, tert-butyl ethers, and Boc groups, as
well as dimethyl acetals were relatively stable to these mild conditions for release of the peptides.
Introduction
solid-phase synthesis.1,2 In the BAL approach, the growing
peptide chain is generally anchored through a backbone amide,
giving easy access to cyclic and C-terminal modified peptides,
e.g., peptide aldehydes (Figure 1).
The backbone amide linker (BAL) methodology has since
the first reports in the mid-1990s become a widely used tool in
The BAL approach has furthermore been extended to the
synthesis of a large variety of amide containing small molecules,
oligosaccharides, and various amine substrates. The BAL
concept was first implemented in a tris(alkoxy)benzyl system,
which allowed release of final products by treatment with
†
Part of this work has been presented in preliminary form in Boas, U.; Jessing,
M.; Christensen, J. B.; Jensen, K. J. Proceedings of the 18th American Peptide
Symposium, Boston, 2003; pp 118-120. Boas, U.; Brask, J.; Christensen, J. B.;
Jensen, K. J. Proceedings of the 27th European Peptide Symposium, Sorrento,
002; pp 50-51. Boas, U. Ph.D. Thesis, University of Copenhagen, 2002.
*
3
2
To whom correspondence should be addressed. Fax: +45 72 34 60 01.
(
K.J.J.) Fax: +45 35 28 23 98.
‡
§
|
⊥
(1) Jensen, K. J.; Alsina, J.; Songster, M. F.; V a´ gner, J.; Albericio, F.;
Barany, G. J. Am. Chem. Soc. 1998, 120, 5441-5452.
(2) For a review, see: Alsina, J.; Jensen, K. J.; Albericio, F.; Barany,
G. Chemistry Eur. J. 1999, 5, 2787-2795.
University of Copenhagen.
Royal Veterinary and Agricultural University.
Danish Institute for Food and Veterinary Research (DFVF).
Contributed equally to this work.
10.1021/jo060687r CCC: $33.50 © 2006 American Chemical Society
6
734
J. Org. Chem. 2006, 71, 6734-6741
Published on Web 08/05/2006

Thiophene Backbone Amide Linkers
FIGURE 2.
FIGURE 1. Schematic depiction of the solid-phase synthesis and
release of peptides using EDOT-based BAL handles.
SCHEME 1a
concentrated trifluoroacetic acid (TFA).1,2 Later analogues, such
as mono- and dialkoxybenzylaldehydes,5 indol, and and
naphthalene-based BAL handles, are somewhat less acid-labile
in the release of peptide substrates.7
4
6
,8
The thiophene backbone amide linkers (T-BAL1, T-BAL2,
T-BAL3) are a new set of handles based on the 3,4-ethylene-
dioxythiophene (EDOT) core structure. EDOT has found wide
9
use as an electron-donating monomer in conducting materials.
The electron-richness of EDOT should make it very suitable as
carbenium ion stabilizing core in acid-labile linkers relying on
the backbone amide linker principle (Figure 2).
In the final handle design all four positions on the thiophene
ring are substituted. The spacer is an alkyl moiety attached
directly to the thiophene ring via a carbon-carbon bond, or
via a thioether linkage. The introduction of a sulfur atom on
the EDOT should provide higher acid lability by additional
stabilization of the carbenium ion (Figure 2).
a
Reagents and conditions: (a) POCl3, DMF, -10 °C, 30 min, 72%; (b)
NIS, AcOH, CHCl3, 25 °C, 16 h or NCS, NaI, acetone, 25 °C, 16 h, 85%;
c) IZn(CH2)2COOMe, Pd2(dba)3, TFP, DMF, 40 °C, 2 h; (d) LiOH (aq),
THF, 25 °C, 2 h, 61%, two steps; (e) HBTU, HOBt, DIPEA, alanine-
(
derivatized polystyrene resin, rt, 2 h, then capping with Ac2O for 2 h.
was observed under the reaction conditions (Scheme 1).13
Alternatively, the R-iodination was carried out with in situ
generated NIS, from N-chlorosuccinimide (NCS) and NaI,14
giving similar yield and purity of the product 2.
Results and Discussion
Synthesis of T-BAL1. In the synthetic sequence, EDOT was
first formylated, to give a more stable (less electron-rich) and
crystalline building block (Scheme 1). The aldehyde 1 was
obtained in 71% yield by Vilsmeier-Haack formylation on
EDOT at -10 °C by slow addition of POCl3, using DMF as
both reactant and solvent.10 Iodination of 1 went smoothly in
Subsequently the spacer was introduced to the EDOT core
by a Negishi C-C cross-coupling reaction between the alkylzinc
reagent of methyl 3-iodopropanoate and 2. DMF was used as
solvent as this solvent generally gives better conversion yields
1
5
8
4% yield at room temperature. Using N-iodosuccinimide (NIS)
with acid-derived organozinc reagents. (3-Carboxymethyl-
propyl)-zinc-iodide was preferred to longer alkyl esters, as a
11,12
activated by acetic acid,
no oxidation of the aldehyde moiety
(
3) Forns, P.; Sevilla, S.; Erra, M.; Ortega, A.; Fernandez, J. C.; Figuera,
(10) For an alternative formylation procedure on EDOT, see: Moha-
nakrishnan, A. K.; Hucke, A.; Lyon, M. A.; Laksmikantham, M. V.; Cava,
M. P. Tetrahedron 1999, 55, 11745-11754.
N. d. l.; Fernandez-Forner, D.; Albericio, F. Tetrahedron Lett. 2003, 44,
6
7
907-6910.
(
4) Swayze, E. E. Tetrahedron Lett. 1997, 38, 8465-8468.
(11) Boas, U.; Dhanabalan, A.; Greve, D. R.; Meijer, E. W. Synlett 2001,
634-636.
(5) Fivush, A. M.; Wilson, T. M. Tetrahedron Lett. 1997, 38, 7151-
154.
(12) For Activation of NBS with acetic acid, see: Wu, R.; Schumm, J.
S.; Pearson, D. L.; Tour, J. M. J. Org. Chem. 1996, 61, 6906-6921.
(13) For an example of a general procedure for iodine-mediated oxidation
of aldehydes to carboxylic acids, see: Yamada, S.; Morizono, D.;
Yamamoto, K. Tetrahedron Lett. 1992, 33, 4329-4332.
(14) Vankar, Y. D.; Kumaravel, G. Tetrahedron Lett. 1984, 25, 233-
236.
(
6) Estep, K. G.; Neipp, C. E.; Stramiello, L. M. S.; Adam, M. D.; Allen,
M. P.; Robinson, S.; Roskamp, E. J. J. Org. Chem. 1998, 63, 5300-5301.
7) Boas, U.; Christensen, J. B.; Jensen, K. J. J. Comb. Chem. 2004, 6,
97-503.
8) Pittelkow, M.; Boas, U.; Jessing, M.; Jensen, K. J.; Christensen, J.
B. Org. Biomol. Chem. 2005, 3, 508-514.
9) Groenendaal, L.; Jonas, F.; Freitag, D.; Pielartzik, H.; Reynolds, J.
R. AdV. Mater. 2000, 12, 481-494.
(
4
(
(
(15) Jackson, R. F. W.; Moore, R. J.; Dexter, C. S. J. Org. Chem. 1998,
63, 7875-7884.
J. Org. Chem, Vol. 71, No. 18, 2006 6735

Jessing et al.
SCHEME 2 a
TABLE 1. Comparison of Cleavage Yields for Leu-Enkephalin on
T-BAL1, o-BAL, and p-BALa
T-BAL1
o-BAL
p-BAL
entry
1a
cleavage conditions
(AAA, %) (AAA, %)19 (AAA, %)
19
95% TFA (aq), 2 h
66
76
71
84
87
82
71
70
89
83
70
64
a
b
b
b
b
2
50% TFA (CH2Cl2), 2 h
5% TFA (CH2Cl2), 2 h
1% TFA (CH2Cl2), 2 h
a
3
a
4
a
For T-BAL1 a resin with a loading of 0.36 mmol/g was used, whereas
b
for o-/p-BAL a resin with a loading of 0.40 mmol/g was used. Cleavage
was conducted over 15 min.
to the procedure by Jung and co-workers.17 Coupling of the
linker molecules to the amino functionalized resin by amide
bond formation was achieved by (benzotriazol-1-ylozy)tris-
a
Reagents and conditions: (a) NBS, CH3CN, rt, 60 h, 91%; (b)
3
8
-mercapto propionic acid, KOH, EtOH, 60 °C, 4 h, then citric acid (aq),
7%; (c) 5-mercapto pentanoic acid, KOH, EtOH, 60 °C, 5 h, then citric
(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) or
acid (aq), 85%.
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methyl-
methanaminium hexafluorophosphate (HBTU) as coupling agent
and DIPEA as base.
result of higher reactivity of the corresponding zinc compound.15
Applying tri-2-furylphosphine (TFP) as ligand for Pd has
previously been shown to increase reaction rates and yields of
the Negishi reaction.16 Finally, 4 equiv of TFP was used together
with 1 equiv of Pd2(dba)3 complex in order to create the highly
Solid-Phase Evaluation of T-BAL1. After initial anchoring
to the solid phase, polystyrene, the T-BAL1-derivatized resin
was applied for the synthesis of unprotected and protected
peptides. Furthermore, it was used for the synthesis of C-
terminal thioesters and ketene dithioacetals. In all cases, the
first building block was anchored by reductive amination.
However, at first this reaction did not go to completion, and
different solvent systems were tried in the anchoring of the first
building block. From the solvent systems used, THF/MeOH/
TMOF (4:1:5) proved to give the highest yield of attached amine
to the T-BAL1 handle for amino acids and primary amines in
general.
0
15
reactive Pd (TFP)2 catalyst during the process. In this way a
new carbon-carbon bond was formed without affecting either
the unprotected aldehyde or the ethyl ester moiety. The
intermediate ester was not isolated, to avoid separation problems.
Instead the ester was hydrolyzed subsequently under mild
conditions by aqueous LiOH, in a one-pot reaction yielding the
carboxylic acid, which could be purified from remaining
iodoaldehyde 2 by extraction. The one-pot reaction afforded
the pure linker 4 in 61% yield as a crystalline compound, stable
to air and light, (Scheme 1).
Then, solid-phase peptide synthesis was conducted under
standard fluorenyloxycarbonyl (Fmoc) conditions, and subse-
quently the peptides were released with different acidic cleavage
mixtures containing TFA, trifluoromethanesulfonic acid (TFM-
Synthesis of T-BAL2 and T-BAL3. With the goal of further
simplifying the synthesis of the handle and increase its acid-
lability, we introduced an exo-cyclic sulfur at the connection
with the spacer. Initial studies on the nucleophilic substitution
on the thiophene R-position with mercaptoalkyl-carboxylic acids
revealed that R-brominated thiophene gave higher yields and
reaction rates than the corresponding iodide, as deiodination of
the thiophene became the prominent reaction. The corresponding
bromide (5, Scheme 2) was synthesized from EDOT aldehyde
18
SA), or AcOH. After 2 h cleavage time the cleavage mixtures
and residual resins were analyzed with HPLC, amino acid
analysis (AAA)/ and magic angle spinning (MAS) NMR.
The immunoreactive pentapeptide Leu-Enkephalin (H-Tyr-
Gly-Gly-Phe-Leu-OH) was chosen as model substrate, as this
peptide has been applied in earlier studies concerning develop-
ment of BAL handles and thus provides comparative informa-
tion. In the solid-phase synthesis of Leu-Enkephalin, cleavage
yields determined by AAA gave comparable yields for the
T-BAL1 handle and the o- and p-BAL handles under high acid
conditions. However, somewhat better cleavage yields were
obtained for T-BAL1 at low acid concentrations (Table 1).
Indications for initial high-yield release of peptides from the
T-BAL1 resin under mild conditions were the low amounts of
peptide released in a second acidolytic release step by treatment
of the peptidyl-resin with TFA/CH2Cl2 (1:1) for 24 h. Similarly,
only small amounts of peptide were released when performing
the second acidolysis with the stronger acid TFMSA.
1
in 85% yield using NBS in CH3CN at room temperature.
Nucleophilic aromatic substitution for introduction of the
spacer moiety was initially conducted as a model experiment,
between 5 and 1-hexanethiol. This reaction was fast, and TLC
showed complete conversion to the corresponding product,
S-((5-formyl-3,4-ethylenedioxy) thiophene-2-yl)-1-thiohexane,
after 30 min at room temperature.
In contrast, the substitution with the mercaptoalkyl-carboxylic
acids proceeded slowly at room temperature; however, by raising
the temperature to 60 °C the reactions took place at a satisfactory
rate. The corresponding thiophene carboxylate adducts were
acidified under mild conditions using dilute citric acid to the
corresponding carboxylic acids 6 and 8 in 87% and 85% yields,
respectively (Scheme 2).
MAS NMR on the peptidyl-resin (data not shown) revealed
a very low content of Leu-Enkephalin present on the resin after
acidolytic treatment with 1% TFA in CH2Cl2, also indicating a
high acid-lability under these conditions for release of the
peptide.
Although 3-mercaptopropionic acid is commercially available,
5-mercaptopentanoic acid (7) was synthesized from the corre-
sponding bromovaleric acid methyl ester and thiourea, similar
(17) Jung, C. M.; Kraus, W.; Leibnitz, P.; Pietzsch, H. J.; Kropp, J.;
Spies, H. Eur. J. Inorg. Chem. 2002, 1219-1225.
(18) No cleavage yields are reported for cleavage with TFMSA as they
did not give higher yields than for similar concentrations of TFA.
(
16) For a review on TFP in transition-metal-mediated organic synthesis,
see: Andersen, N. G.; Keay, B. A. Chem. ReV. 2001, 101, 997-1030.
6736 J. Org. Chem., Vol. 71, No. 18, 2006

Thiophene Backbone Amide Linkers
SCHEME 3 a
a
Reagents and conditions: (a) -Cl H3N-CH2-C(EtS)3, NaBH3CN, DMF, 16 h; (b) Fmoc-Gly-OH, DIPCDI, CH2Cl2/DMF 9:1, 16 h, then SPPS using
+
Fmoc strategy; (c) TFA/H2O (19:1), 2 h, 65% or HOAc/H2O (1:1), 2 h, 40%; (d) TFA/CH2Cl2 (1:1), 2 h, 63%.
SCHEME 4. Proposed Mechanism for â-Elimination
Leading to Decreased Loading during Solid-Phase Synthesis
on the T-BAL2 Handle
For further test of the methodology with several di- and
tripeptides with different C-terminal functionalities, low-acid
conditions combined with quenching of the acidic mixture with
base released the protected peptides without affecting acid-labile
protecting groups, e.g., the Boc- or the tert-butylester group.
The possibility to cleave off peptides having a thioester group
is intriguing as these activated substrates may be applied for
subsequent synthesis of polypeptide or protein substrates by
Kent segmental coupling strategy. A tripeptide trithioortho ester
(11) was synthesized on the T-BAL1-derivatized solid phase,
where the trithioortho ester group serves as a base-stable but
acid-labile masking group for the C-terminal thioester and ketene
dithioacetal functionalities. The trithioortho ester was cleaved
from the resin in 63% yield either as the thioester (12) using
9
5% aqueous TFA or, again, in 63% yield as the ketene
dithioacetal (13) using anhydrous conditions, e.g., TFA/CH2-
Cl2 (1:1) (Scheme 3).20
It is quite remarkable that peptide thioesters could also be
released in 40% yield from T-BAL1 with 50% aqueous acetic
acid. This hyper acid-lability of the T-BAL1 handle appears to
be specific for the peptide trithioetho ester, in which steric
congestion when attached to the resin facilitates the cleavage
process.
The dipeptide aldehyde Fmoc-Lys-Gly-H was synthesized and
released in 62% yield by TFA/CH2Cl2 (1:1). In contrast, when
the peptide was released with 1% TFA in CH2Cl2, the acid-
labile C-terminal dimethyl acetal, precursor of the aldehyde,
mainly remained intact. Furthermore, we investigated the
possibility to release more basic functionalities (e.g., amines)
directly from the T-BAL1 handle.21 We found that the aliphatic
primary amine Fmoc-HN(CH2)2-NH2 and the secondary amine
FmocHN(CH2)2NHCH2Ar could be released by TFA/CH2Cl2
(
1:1) after 2 h at 60 °C in moderate yields of 27% and 37%,
respectively.
Solid-Phase Evaluation of T-BAL2 and T-BAL3. Upon
performing solid-phase synthesis of Leu-Enkephalin on T-
BAL2, decreasing yields of released peptide were observed after
each coupling. An explanation for this observation may be the
susceptibility of this linker to â-elimination when subjected to
the basic conditions required for cleavage of the Fmoc group
after each solid-phase coupling (Scheme 4). Because of this
considerable drawback, solid-phase synthesis based on this linker
was not investigated further.
To investigate the initial reductive amination and acylation
steps in the synthesis of the peptide backbone, several dipeptide
sequences were synthesized and released from the T-BAL3
linker. For the reductive amination step the solvent system
TMOF/MeOH (4:1) resulted in significantly better reaction
yields compared to 1% acetic acid in MeOH. TMOF is a well-
known condensation reagent for the formation of imines. In
addition, higher temperatures and longer reaction times led to
increasing yields. Shorter reaction times and similar yields were
(
19) Boas, U.; Brask, J.; Christensen, J. B.; Jensen, K. J. J. Comb. Chem.
002, 4, 223-228.
20) Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett. 2003, 5, 2951-
953.
21) For the synthesis of secondary amines on the p-BAL handle, see:
2
(
2
(
Forns, P.; Sevilla, S.; Erra, M.; Ortega, A.; Fern a´ ndez, J.-C.; de la Figuera,
N.; Fern a´ ndez-Forner, D.; Albericio, F. Tetrahedron Lett. 2003, 44, 6907-
6
910.
J. Org. Chem, Vol. 71, No. 18, 2006 6737

Jessing et al.
yield (%)
TABLE 2. Reaction Conditions and Yields for the Reductive Amination of T-BAL3a
peptide
additives and solvents
reaction conditions
Fmoc-Ala-Gly-OH
Fmoc-Ala-Gly-OH
Fmoc Ala-Gly-OH
Fmoc Ala-Gly-OH
Fmoc Ala-Gly-OH
Fmoc Ala-Gly-OH
Fmoc-Ala-Leu-OH
Fmoc-Ala-Leu-OH
Fmoc-Ala-Leu-OH
Fmoc-Ala-Leu-OH
Fmoc-Ala-Leu-OH
AcOH/MeOH (1:99)
AcOH/MeOH (1:99)
TMOF/MeOH (4:1)
microwave, 60 °C, 10 min
microwave, 60 °C, 2 × 10 min
microwave, 60 °C, 2 × 10 min
microwave, 60 °C, 2 × 10 min
microwave, 60 °C, 10 min
microwave, 60 °C, 10 min
microwave, 60 °C, 10 min
microwave, 60 °C, 10 min
microwave, 60 °C, 10 min
heater shaker, 60°C, 16 h
heater shaker, 100°C, 1 h
71
75
71
68
70
69
10
13
11
32
37
HOAc/TMOF/MeOH (1:80:20)
1 equiv of Sc(Otf)3, MeOH
i
1 equiv of Ti(O Pr)4, MeOH
TMOF/MeOH (4:1)
1 equiv of Sc(Otf)3, MeOH
i
1 equiv of Ti(O Pr)4, MeOH
TMOF/MeOH (4:1)
TMOF/MeOH (4:1)
a
Conditions for acylation of the secondary amine: Fmoc-Ala-OH (10 equiv) and DIPCDI (5 equiv) in CH2Cl2/DMF (9:1), rt., 2 h, repeated once; loading
determined by Fmoc quantification.
TABLE 3. Cleavage (Release) Yields from
Conclusion
H-Tyr-Gly-Gly-Phe-(T-BAL3-Ile-PS)LeuNH2 Analyzed by Amino
Acid Analysis Using Ile as Internal Reference Amino Acid (IRAA)a
Three new BAL type handles based on the commercially
available electron-rich building block EDOT have been prepared
by a three step synthetic sequence. The new T-BAL handles
have proven useful as a set of very acid-labile linkers utilized
in the BAL solid-phase methodology. Generally, the new linkers
are more acid-labile than the original trialkoxybenzyl based BAL
handles. The new handles were used for synthesis of protected
and partly protected peptides carrying acid-labile functionalities
such as dimethyl acetals, tert-butyl esters, and tert-butyl ethers.
Thioesters or ketene dithioacetals could, depending on the
cleavage conditions be released from a T-BAL1 resin. The
T-BAL handles are the first reported handles where peptide
thioesters could be released under extremely mild conditions
using aqueous acetic acid. This can possibly be rationalized by
ground-state destabilization of the T-BAL1 anchored peptide
due to the bulky trithioortho ester moiety. All of the peptides
synthesized were released in high purity according to HPLC.
Leu-Ile Phe-Ile Leu-Ile Phe-Ile
cleavage
conditions
ratio
before before
ratio
ratio
after
ratio
after
yield
(%)
entry
1
2
3
TFA/H2O (95:5)
TFA/CH2Cl2 (5:95)
TFA/CH2Cl2 (1:99)
0.71
0.71
0.71
0.73
0.73
0.73
0.11
0.17
0.71
0.044 85-94
0.16
0.73
76-78
0
a
Cleavage (release) yield is here defined as (before - after)/before ×
1
“
00%, where “before” is the peptidyl-resin prior to acid treatment, and
after” is subsequent to acid treatment.
achieved using microwave heating (Table 2). As a general trend,
the reductive amination yields decreased with increasing bulki-
ness of the C-terminal amino acid, where dipeptides containing
a C-terminal leucine were released in low to moderate yields.
Addition of 1 equiv. of a Lewis acid such as Sc(OTf)3 or Ti-
(
OiPr)4 did not result in improved yields. Applying other
additives such as the condensation reagents TMS-Cl or Ph3-
PBr2 did not increase the effectiveness of the preceding imine
formation either.
T-BAL2 and T-BAL3, which contain an exocyclic thioether
as bridging point to the spacer, were synthesized via a more
convenient route, well-suited for large scale synthesis compared
to the T-BAL1 handle. However, upon performing solid-phase
peptide synthesis (SPPS) on the T-BAL2 handle the yields of
released peptide decreased rapidly during the course of the
synthesis, most likely as a result of â-elimination. For the
T-BAL3 handle, the bulkiness of the first amino acid building
block had a large effect on the yield of the initial reductive
amination, where Leu gave significantly lower yields. However,
less bulky amino acids were incorporated in satisfactory yields.
Investigations by AAA on the acid-lability of the T-BAL3
handle in the release of Leu-Enkephalin showed a somewhat
lower acid-lability of T-BAL3 in comparison to the initial
T-BAL1 handle. In addition, the release of a peptide less
sterically congested around the linkage to T-BAL3 was studied,
conforming in this case the effect of (lack of) steric bulk on
release of peptide from the resin. Interestingly, this study
revealed that steric factors (i.e. steric relief) during the release
of the peptide may affect the yield of the released peptide,
showing a tendency of lower release of less bulky peptides.
However a more thorough study concerning the effect of
sterically congested amino acids at the anchoring site will have
to be performed, to show if the lower release yields are a
common feature in the release of less sterically congested
peptides from BAL-based handles. However, in general both
T-BAL1 and T-BAL3 show somewhat higher acid-labilities in
comparison to previously reported BAL handles based on a
homo-aromatic core structures, making this novel class of
Generally, the use of Tentagel resin resulted in overall better
cleavage yields in comparison to a pure polystyrene resin.
Finally, upon finding the optimal conditions for the reductive
amination and acylation steps, the effectiveness of the release
of Leu-Enkephalin from the T-BAL3 handle was investigated
by AAA. These investigations showed that high acid conditions
(
95% aqueous TFA) led to release of the peptide from T-BAL3
in excellent yields (85-94%). Low acid conditions (5% TFA
in CH2Cl2) released the peptide in good yields (76-78%),
whereas no release of peptide from the resin could be measured
upon employment of a 1% TFA in CH2Cl2 cleavage mixture
(Table 3).
Interestingly, release from the resin of the peptide H-Tyr-
Gly-Gly-Phe-Leu-Gly-Gly-OH, which is less sterically con-
gested around the anchoring point to the handle, resulted in
somewhat lower yields of peptide. In this study the T-BAL1
and T-BAL3 had comparable acid-labilities, releasing 45-55%
peptide under high acid conditions (95% TFA in CH2Cl2 or 95%
aqueous TFA). In these investigations modest yields (8-12%)
of peptide were released under low-acid conditions (1%TFA
in CH2Cl2) from both handles. The lower yields in the release
of this sequence, at 5% and 95% TFA, indicate that steric relief
may play an important role in the release of the peptide from
these thiophene based linkers. However, it was rewarding to
observe that for this particular peptide, some release occurred
with 1% TFA.
6738 J. Org. Chem., Vol. 71, No. 18, 2006

Thiophene Backbone Amide Linkers
1
thiophene based linkers a promising motif for highly acid labile
BAL handles.
acetate-hexane gave the product as yellow crystals. H NMR (500
MHz, d
(
6
-DMSO) δ 9.76 (s, 1H), 4.38 (m, 2H), 4.29 (m, 2H), 2.86
3
3 13
t, J ) 7.3 Hz, 2H), 2.54 (t, J ) 7.3 Hz, 2H); C NMR (125
MHz, d
6
-DMSO) δ 179.5, 173.6, 149.5, 139.0, 129.3, 114.9, 66.2,
Experimental Section
6
5.0, 34.0, 22.4. Anal. Calcd (C10H10SO (242.26)): C 49.57, H
5
-(3,4-Ethylenedioxythiophene) carbaldehyde (1).23 3,4-Eth-
ylenedioxythiophene (50 g, 0.35 mmol) was dissolved in dry DMF
200 mL). The mixture was cooled to -10 °C (ice-ethanol bath)
and POCl (33 mL, 0.36 mmol) was added dropwise (15 min) in
4.17, S 13.23. Found: C 49.10, H 4.08, S 12.98. MS (FAB
+
) found
2
+
242.99 (MH ).
(
2-Bromo-(3,4-ethylenedioxythiophene)-5-carbaldehyde (5).
Compound 1 (4.04 g, 23.7 mmol) was suspended in dry acetonitrile
(100 mL) and cooled to 0 °C. NBS (4.36 g, 26.0 mmol), 1.1 equiv,
was added and the mixture was stirred for 60 h at room temperature,
shielded from light and under nitrogen. The color changed from
yellow to purple. The mixture was transferred with 150 mL ofethyl
acetate to a separation funnel, washed with 10% aqueous Na
(2 × 200 mL), saturated Na (2 × 200 mL) and water (2 ×
200 mL), dried with MgSO , and evaporated in vacuo. Recrystal-
3
the cold. The mixture stirred 1 h at -10 °C, ice water (500 mL)
was added and the mixture stirred overnight at room temperature.
The aldehyde was filtered off, dissolved in CH
dried (Na SO ). The CH Cl filtrate was eluted through a short silica
plug” to remove colored byproducts, giving the dry product as
2 2
Cl (500 mL) and
2
4
2
2
“
2 3
CO
1
0
slightly tanned crystals. Yield 42 g (71%); mp 145-146 °C (lit.
2 2 3
S O
1
1
4
1
42 °C); H NMR (500 MHz, CDCl
.37 (m, 2H), 4.28 (m, 2H); 13C NMR (125 MHz, CDCl
49.2, 142.5, 119.2, 111.4, 66.0, 65.1. Anal. Calcd (C
3
) δ 9.91 (s, 1H), 6.80 (s, 1H),
) δ 180.7,
SO
4
lization twice from ethanol (60 mL) yielded 5.35 g (91%) of the
3
1
H
7 6
3
bromide as yellow needles. Mp 138-140 °C (decomp); H NMR
3
(
1
170.19)): C 49.40, H 3.56, S 18.84. Found: C 49.41, H 3.43 S
8.78. MS (FAB ) found 170.96 (MH ).
(300 MHz, CDCl
MHz, CDCl
Calcd (C
3
) δ 9.83 (s, 1H), 4.36 (m, 4H); 1 C NMR (75
+
+
3
) δ 179.0, 147.9, 140.4, 118.8, 102.0, 65.5, 65.1. Anal.
BrO S (249.03)): C 33.75, H 2.02, S 12.87. Found:
2-Iodo-(3,4-ethylenedioxythiophene) 5-carbaldehyde (2). Com-
7
H
5
3
+
+
pound 1 (2.0 g, 11.8 mmol) was suspended in dry chloroform (10
mL), and glacial acetic acid (10 mL) was added followed by NIS
C 33.48, H 1.79, S 13.98; MS (FAB ) found 248.94 (M ).
S-((5-Formyl-3,4-ethylenedioxy)thiophene-2-yl)-3-thiopropi-
onic Acid (6). KOH (1.14 g, 20.3 mmol), 3 equiv, and 3-mercap-
topropionic acid (0.64 mL, 7.34 mmol), 1.2 equiv, were dissolved
in ethanol (50 mL) and stirred for 10 min. Compound 5 (1.59 g,
6.38 mmol) was added initially giving a yellow solution that turned
(2.9 g, 13.0 mmol), giving a deep purple color of the mixture. The
mixture was shielded from light and stirred overnight at room
temperature, changing to a red suspension. Ethyl acetate (50 mL)
was added and the organic layer was washed with 10% Na
aq) (2 × 50 mL), Na (sat.) (2 × 50 mL), and water (2 × 50
mL). The residue was dried (Na SO ), evaporated in vacuo and
2 3
CO
(
S
2 2
O
3
orange upon stirring under nitrogen for 4 h at 60 °C. CH
mL) was added and the mixture was extracted with water (150 mL)
and saturated Na (50 mL). The aqueous layer was backwashed
with CH Cl (100 mL) to remove residual bromide. The aqueous
layer was acidified with citric acid (1.59 g, 7.57 mmol), and the
solution was stirred for 10 min. CH Cl (100 mL) was added and
the mixture was stirred for another 40 min. The aqueous layer was
extracted with additional CH Cl (100 mL). The organic phase was
dried with Na SO and evaporated in vacuo. Yield 1.53 g (87%)
as light yellow needles. Mp 109-113 °C (decomp); H NMR (300
MHz, CDCl ) δ 11.0-10.2 (bs, 1H), 9.82 (s, 1H), 4.34 (m, 4H),
2 2
Cl (150
2
4
recrystallized from ethanol to yield 2.9 g (84%) of the product as
2 2 3
S O
1
a bright yellow crystalline. Mp 156-157 °C; H NMR (500 MHz,
2
2
3 3
CDCl )
) δ 9.78 (s, 1H), 4.36 (m, 4H); 13C NMR (75 MHz, CDCl
δ 1778.8, 147.0, 144.4, 123.4, 110.9, 65.4, 65.1. Anal. Calcd (C
SO I (296.08)): C 28.39, H 1.71, S 10.83. Found: C 28.75, H
.48 S 10.44. MS (FAB ) found 296.83 (MH ).
-(5-Formyl-3,4-ethylenedioxy)thiophen-2-yl) Propionic Acid
4). Zinc dust (0.907 g, 13.6 mmol) was suspended in dry DMF (3
7 5
H -
2
2
3
+
+
1
2
2
3
2
4
1
(
mL) and activated by addition of 1.2-dibromoethane (59 µL),
followed by stirring 30 min at 60 °C, after cooling to room
temperature. TMS-Cl (18 µL) was added and the mixture was
stirred vigorously for 30 min at room temperature. The mixture
3
3
3
13
3.11 (t, J ) 7 Hz, 2H), 2.72 (t, J ) 7 Hz, 2H); C NMR (75
MHz, CDCl ) δ 179.3, 177.0, 148.2, 142.7, 120.7, 119.1, 65.3, 64.9,
34.4, 30.9. Anal. Calcd (C10 (274.32)): C 43.78, H 3.67,
S 23.38; found C 43.52, H 3.45, S 22.80. MS (FAB ) found 273.97
3
10 5 2
H O S
24
+
was cooled on an ice bath and the iodoester 3 (0.53 g, 2.3 mmol)
was added dropwise in the cold; after 10 min the ice bath was
removed. The mixture was stirred for 30 min at room temperature.
generating the alkylzinc reagent (TLC (ethyl acetate) shows
quenching of the iodide). The slurry was centrifuged and the
supernatant was transferred to a mixture of compound 2 (0.50 g,
+
+
(M ) and 274.98 (MH ).
5-Mercapto-pentanoic Acid (7). A mixture of thiourea (1.35
g, 17.7 mmol), 1.5 equiv, and 5-bromo-pentanoic acid ethyl ester
(1.9 mL, 12.0 mmol) was refluxed in EtOH (25 mL) for 20 h. The
solvent was removed in vacuo and 7.5 M NaOH (aq) (25 mL, 188
mmol), 15 equiv, was added. The mixture was stirred for an
additional 16 h at 90 °C, under nitrogen. It was then cooled on an
1
.7 mmol), Pd
mmol) in dry DMF (2 mL); The mixture heats to approximately
0 °C spontaneously. The dark orange mixture was stirred for 2 h
2 3
(dba) (15 mg, 0.014 mmol) and TFP (15 mg, 0.061
4
ice bath and 2M H
organic product was extracted with CH
with MgSO . Evaporation in vacuo gave a colorless oil in
2
SO
4
was added slowly under stirring. The
at 40 °C. HPLC showed quantitative conversion to the ester adduct.
LiOH (0.71 g, 17.00 mmol in 12 mL water) was added, and the
mixture stirred for 2 h at room temperature, hydrolyzing the ester.
Water (100 mL) was added and the polar layer was extracted with
2
Cl
2
(2 × 100 mL), dried
4
quantitative yield. The product was used at once in the next reaction
as formation of disulfide was observed by prolonged storage, even
1
CH
2
Cl
2
(2 × 100 mL). Concentrated aqueous HCl (2.6 mL, 26
under nitrogen. H NMR (300 MHz, CDCl
3
) δ 11.3-10.2 (br. s,
) 7 Hz, J ) 8 Hz, 2H), 2.37 (t, J ) 7 Hz,
25
3
3
3
mmol) was added and the water layer was quickly extracted with
1H), 2.54 (dt/q, J
2H), 1.80-1.59 (m, 4H), 1.35 (t, J ) 7 Hz,1H).
1
2
3
ethyl acetate (1 × 150 mL). The ethyl acetate layer was washed
with brine, dried (Na
2
4
SO ), and evaporated in vacuo. The residue
S-((5-Formyl-3,4-ethylenedioxy)thiophene-2-yl)-5-thiopen-
tanoic acid (8). KOH (1.77 g, 31.5 mmol), 3 equiv, and freshly
made 5-mercaptopentanoic acid 7 (1.42 g, 10.6 mmol), 1.0 equiv,
were dissolved in 50 mL of ethanol and stirred for 10 min.
Compound 5 (2.65 g, 10.6 mmol) was added initially giving a
yellow solution. After stirring under nitrogen for 5 h at ∼ 60 °C,
the solution had turned orange. Water (100 mL) and saturated
was triturated from diethyl ether to give 0.250 g (61%) of the
product as a curry tanned powder. Reprecipitation from ethyl
(
22) Knochel, P.; Yeh, M. C. P.; Berk, S. C.; Talbert, J. J. Org. Chem.
988, 53, 2390-2392.
23) The correct IUPAC nomenclature for this compound is 2,3-dihydro-
1
(
thieno [3,4-b][1,4] dioxine-5-carbaldehyde, but for this and the following
compounds the more brief “EDOT-nomenclature” will be applied.
Na
with CH
equiv, was added and the solution was stirred for 10 min. CH
2
S O
2 3
(50 mL) was added and the aqueous layer was washed
Cl
(2 × 120 mL). Citric acid (2.67 g, 12.7 mmol), 1.2
Cl
2
2
(
24) Tok, J. B. H.; Cho, J.; Rando, R. R. Tetrahedron 1999, 55, 5741-
758.
25) Alternatively, 10% aqueous citric acid can be used in order to give
a milder acidification of the reaction mixture.
2
2
5
(
(2 × 100 mL) was added and the mixture stirred another 40 min
before collection of the organic phase. The organic phase was
J. Org. Chem, Vol. 71, No. 18, 2006 6739

Jessing et al.
backwashed with water (2 × 100 mL), dried with (Na
2
SO
4
) and
95%TFA (aq); (B) TFA/CH
2
Cl
2
(1:1); (C) 5% TFA/CH
2 2
Cl ; (D)
evaporated in vacuo. Yield was 3.12 g (97%) of the product as a
1% TFA/CH Cl . The resins were washed with CH
2
2
2
Cl and MeOH
2
yellow powder containing a little disulfide. A part of the product,
and dried in vacuo. The content of residual peptide on the resins
was determined by AAA, using the uncleaved fully derivatized
peptidyl resin as reference.
2.13 g, was dissolved in MeOH (10 mL/g compound) and water
was added (100 mL) giving a milky slurry. Upon standing overnight
crystals precipitated out. The crystals were filtered with suction,
Fmoc-Phe-Gly-Gly-SEt (12). To the T-BAL1-derivatized resin
washed with water and dried. Yield 1.80 g (85%) of light yellow
9 (0.200 g, 0.074 mmol) were added H-Gly(SEt) ‚HCl (0.037 g,
3
1
needles. Mp 89-91 °C; H NMR (300 MHz, CDCl
3
) δ 11.6-10.8
0.139 mmol), NaBH CN (0.023 g, 0.369 mmol) and THF/MeOH
3
(bs, 1H, CO
2
H), 9.77 (s, 1H, CHO), 4.34 (m, 4H, OCH
2
CH O),
2
13
(4:1) (2 mL), and the mixture was shaken for 24 h, washed with
3
3
2
.92 (t, J ) Hz, 2H), 2.38 (t, J ) 7 Hz, 2H), 1.75 (m, 4H);
NMR (75 MHz, CDCl ) δ 179.0, 178.9, 148.2, 141.7, 122.9, 118.4,
5.3, 64.8, 35.8, 33.4, 28.8, 23.5. Anal. Calcd (C12
302.37)): C 47.67, H 4.67, S 21.21. Found: C 48.02 H 4.61 S
C
DMF (8 × 10 mL), CH Cl (4 × 10 mL), and MeOH (2 × 10
2
2
3
mL), and dried in vacuo. Fmoc-Gly-OH (0.442 g, 1.486 mmol)
was dissolved in DMF/CH Cl (1:9) (6 mL), and DIPCDI (0.092
6
(
15 5 2
H O S
2
2
g, 0.726 mmol) was slowly added under continuous shaking. The
slurry was then added to the derivatized resin (0.019 mmol) and
+
+
+
2
1.39. MS (FAB ) found 302.08 (M ) and 303.08 (MH ).
Anchoring T-BAL1 to Aminomethylated PS Resin via Ala
as Internal Reference Amino Acid (IRAA) (9). Aminomethylated
polystyrene resin (1.929 g, 0.694 mmol) was washed with DMF
shaken for 20 h, washed with DMF (8 × 10 mL), CH
2
Cl
2
(4 × 10
mL), and MeOH (2 × 10 mL), and dried in vacuo. The Fmoc-
protected resin was treated with piperidine/DMF (1:4) (3 × 5 mL)
for 2 × 2 min and 1 × 20 min, then washed with DMF (5 × 10
(
2
3 × 10 mL) and CH
2
Cl
2
(3 × 10 mL). Fmoc-Ala-OH (0.889 g,
.857 mmol), HBTU (1.01 g, 2.655 mmol) and 1-hydroxybenzo-
mL), CH
2
Cl
2
(4 × 10 mL), and MeOH (1 × 10 mL) and dried in
triazol (HOBt) (0.393 g, 2.907 mmol) were dissolved in DMF (7
mL). DIPEA (0.818 g, 6.33 mmol) was added and the mixture was
preactivated for 5 min and added to the resin. The mixture was
then shaken for 2 h and afterward washed with DMF (4 × 10 mL),
vacuo. Fmoc-Phe-OH (0.117 g, 0.302 mmol), HBTU (0.108 g,
0.285 mmol), and HOBt (0.041 g, 0.300 mmol) were dissolved in
DMF (3 mL). DIPEA (0.082 g, 0.631 mmol) was added, and the
mixture was preactivated for 5 min. The mixture was added to the
derivatized resin (0.074 mmol), shaken for 1 h, washed with DMF
CH
2
Cl
2
(2 × 10 mL), and MeOH (10 mL) and dried in vacuo.
Residual aminopolystyrene groups were capped with 20% acetic
anhydride for 1 h. The Fmoc-Ala-functionalized PS resin was
treated with piperidine/DMF 1:4 (3 × 12 mL) for 2 × 2 min and
(8 × 10 mL), CH
dried in vacuo. To portions of the derivatized resin (3 × 20 mg)
was added (A) TFA/H O (19:1), (B) TFA/CH Cl (1:19), or (C)
AcOH/CH Cl (1:1) (1 mL), after 2 h it was washed with the
cleavage mixture (2 × 1.5 mL) and CH Cl (4 × 1.5 mL) and
evaporated. HPLC t ) 8.3 min (A, 100%; B, 75%; C, 81%), TOF
2
Cl
2
(4 × 10 mL), and MeOH (2 × 10 mL), and
2
2
2
1
×
× 20 min and then washed with DMF (8 × 10 mL), CH
10 mL), and MeOH (2 × 10 mL) and dried in vacuo.
2
Cl
2
(4
2
2
2
2
Compound 4 (0.165 g, 0.681 mmol), HBTU (0.262 g, 0.690
R
+
+
mmol) and HOBt (0.096 g, 0.708 mmol) were dissolved in DMF
5 mL), DIPEA (0.176 g, 1.360 mmol) was added and the mixture
was preactivated for 5 min. The mixture was then added to the
Ala-functionalized PS resin (0.347 mmol), shaken for 16 h, washed
ES-MS calcd (C30
H
31
N
3
5
O S (545.65)), found 546.21 (MH ) and
+
(
568.19 (M + Na ).
Fmoc-Phe-Gly-NHCHdC(SEt)
tized resin 11 was added TFA/dry CH
h it was washed with the cleavage mixture (2 × 2 mL) and CH
(3 × 1 mL) and evaporated. HPLC t
Cl ) 8.3 min (8%) for the
thioester and t ) 9.3 min (92%) for the ketene dithio acetal
35 3 4 2
protected peptide. TOF ES-MS calcd (C32H N O S (589.77)),
2
(13). To 20 mg of the deriva-
2
Cl (1:99) (1 mL). After 2
2
with DMF (4 × 10 mL), and then shaken with Ac
2
O (0.387 g,
2
-
3
.793 mmol) in DMF (4 mL) for 1 h. The resin was washed with
2
R
DMF (8 × 10 mL), CH
2
Cl
2
(4 × 10 mL), and MeOH (2 × 10 mL)
R
+
and dried in vacuo.
+
+
Fmoc-Tyr-Gly-Gly-Phe-Leu-OH (10). To the T-BAL1-deriva-
tized resin 9 (0.153 g, 0.055 mmol) were added H-Leu-OtBu‚HCl
0.132 g, 0.590 mmol), NaBH CN (0.042 g, 0.675 mmol) and THF/
3
found 589.00 (M ) and 612.20 (M + Na ).
Fmoc-Lys(Boc)-Gly(OMe) (14). To the T-BAL1-derivatized
resin 9 (0.156 g, 0.056 mmol) in THF/MeOH/TMOF (4:1:5)
containing 1% AcOH (2.5 mL) were added H-Gly(OMe) (0.158
g, 1.50 mmol) and NaCNBH (0.072 g, 1.15 mmol), and the mixture
was shaken for 16 h, washed with DMF (8 × 10 mL), CH Cl (4
× 10 mL), and MeOH (2 × 10 mL), and dried in vacuo. Fmoc-
Lys(Boc)-OH (0.540 g, 1.15 mmol) was dissolved in DMF/CH
Cl (1:9) (10 mL), and DIPCDI (0.135 g, 1.07 mmol) was slowly
2
(
MeOH/TMOF (4:1:5) (2 mL) and the mixture was shaken for 16
h, washed with DMF (8 × 10 mL), CH Cl (4 × 10 mL), and
MeOH (2 × 10 mL), and dried in vacuo. Fmoc-Phe-OH (0.438 g,
.130 mmol) was dissolved in DMF/CH Cl (1:9) (8 mL) and N,N′-
2
2
2
3
2
2
1
2
2
diisopropylcarbodiimide (DIPCDI) (0.157 g, 1.240 mmol) was
slowly added under continuous shaking. The slurry was then added
to the derivatized resin (0.055 mmol), shaken for 16 h, washed
2
-
2
added under continuous shaking. The slurry was then added to the
acetal-derivatized resin (0.047 mmol), shaken for 18 h, and washed
with DMF (8 × 10 mL), CH
0 mL), and dried in vacuo. The Fmoc-protected resin was treated
with piperidine/DMF (1:4) (3 × 5 mL) for 2 × 2 min and 1 × 20
Cl
2
Cl
2
(4 × 10 mL), and MeOH (2 ×
1
with DMF (8 × 10 mL), CH
10 mL) followed by air-drying.
The derivatized resin was divided into portions (3 × 20 mg) to
which was added (A) TFA/H O (19:1), (B) TFA/CH Cl (1:1), or
(C) TFA/CH Cl (1:99) (1 mL). After 2 h the mixture was washed
with the cleavage mixture (2 × 1.5 mL) and CH Cl (2 × 2 mL).
For B the washes were neutralized with 10% NaHCO (aqueous)
and evaporated. HPLC t ) 6.8 min (A, 100%; B, 89%; C, 60%)
for the unprotected peptide and t ) 8.3 min (C, 40%) for the acetal-
and Boc-protected peptide). ES-MS calcd (C30
2
Cl
2
(4 × 10 mL), and MeOH (2 ×
min, then washed with DMF (4 × 10 mL), CH
2
2
(2 × 10 mL),
and MeOH (1 × 10 mL), and dried in vacuo. Subsequent
derivatization of the dipeptidyl resin was performed using standard
SPPS protocol. Coupling reactions were carried out as follows:
Fmoc-AA-OH (4 equiv) was dissolved in DMF and preactivated
for 5 min with HBTU (3.8 equiv), HOBt (4 equiv), and DIPEA
2
2
2
2
2
2
2
3
R
(
7.8 equiv) and transferred to a filter syringe with the derivatized
R
+
resin (1 equiv based on loading measurement). Before the final
Fmoc deprotection, the loading of the peptidylresin was calculated
to 0.1210 mmol/g according to the UV absorption from the
dibenzofulvene-piperidine complex. The Leu-Enkephalin deriva-
tized resin was subsequently subjected to various acidolytic
41 3 7
H N O (555.66)),
+
2
found 392.0 (MH - H O (imine)).
Optimized Reductive Amination of T-BAL3 Using Micro-
waves. H-Leu-OtBu (20 equiv) or H-Gly-OtBu (20 equiv) and
NaBH CN (20 equiv) were suspended in TMOF/MeOH (4:1) and
3
conditions where the yields of released pentapeptide were measured
added to the T-BAL3-derivatized resin. The mixture was irradiated
with microwaves at 60 °C for 10 min. The solvents were removed
by suction, and the resin washed with DMF (3 times), DCM (3
times), and DMF (3 times).
+
by AAA. TOF ES MS calcd (C43
H N
47 5
O
9
(777.86)), found 778
+
(M ).
Acidolytic Cleavage of Leu-Enkephalin from T-BAL1 (Table
1
×
). Leu-Enkephalin-derivatized resin was divided into portions (5
20 mg), where acidolysis was carried out for 2 h using (A)
Acidolytic Cleavage of Leu-Enkephalin from T-BAL3. Ini-
tially, the protected peptidyl resin was Fmoc-deprotected with
6740 J. Org. Chem., Vol. 71, No. 18, 2006

Thiophene Backbone Amide Linkers
piperidine.DMF (1:4). Small samples of Leu-Enkephalin-derivatized
resin (approximately 10 mg) were treated with various acidic
cleavage mixtures for 2 h at room temperature. The supernatant
from each cleavage experiment was removed by suction, and the
assistant Jette Petersen is gratefully acknowledged for assistance
in performing AAA.
Supporting Information Available: Experimental general
1
13
remarks. Experimental procedure for compound 3. H and C NMR
data on compound 5. HPLC data (265 nm) on crude Fmoc-Tyr-
Gly-Gly-Phe-Leu-OH, crude Fmoc-Gly-Leu-OtBu, crude Fmoc-
residual resin washed with CH
analyzed by AAA.
2 2
Cl . The residual resins were
2
Phe-Gly-Gly-SEt, and crude Fmoc-Phe-Gly-NHCHdC(SEt) re-
Acknowledgment. J.B.C. and U.B. thank the Lundbeck
foundation and Leo Pharma foundation for financial support.
M.J. acknowledges a student grant from Novo Nordisk. K.J.J.
acknowledges financial support from the Danish Natural Science
Research Council. Dr. L. Groenendaal is acknowledged for his
advice regarding handling and purchasing of EDOT. Laboratory
leased from T-BAL1 resin. Cleavage yields of H-Tyr-Gly-Gly-Phe-
Leu-Gly-Gly-OH from T-BAL1 and T-BAL3 resins based on amino
acid analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO060687R
J. Org. Chem, Vol. 71, No. 18, 2006 6741