Aryldithioethyloxycarbonyl (Ardec): A new family of amine protecting groups removable under Mild reducing conditions and their applications to peptide synthesis

Article abstract of DOI:10.1002/chem.200501538

The development of phenyl-dithioethyloxycarbonyl (Phdec) and 2-pyridyldithioethyloxycarbonyl (Pydec) protecting groups, which are thiollabile urethanes, is described. These new disulfide-based protecting groups were introduced onto the ε-amino group of L-

Full text of DOI:10.1002/chem.200501538

FULL PAPER
DOI: 10.1002/chem.200501538
Aryldithioethyloxycarbonyl (Ardec): A NewFamily of Amine Protecting
Groups Removable under Mild Reducing Conditions and Their Applications
to Peptide Synthesis**
Milaine Lapeyre,[a] JØrôme Leprince,[c] Marc Massonneau,[d] Hassan Oulyadi,[b]
Pierre-Yves Renard,[a] Anthony Romieu,*[a] Gerardo Turcatti,[e] and Hubert Vaudry[c]
Abstract: The development of phenyl-
dithioethyloxycarbonyl (Phdec) and 2-
pyridyldithioethyloxycarbonyl (Pydec)
protecting groups, which are thiol-
labile urethanes, is described. These
new disulfide-based protecting groups
were introduced onto the e-amino
group of l-lysine; the resulting amino
acid derivatives were easily converted
into Na-Fmoc building blocks suitable
for both solid- and solution-phase pep-
tide synthesis. Model dipeptide-
Ardec groups both in aqueous and or-
ganic media. Phdec and Pydec were
found to be cleaved within 15 to
30 min under mild reducing conditions:
i) by treatment with dithiothreitol or b-
mercaptoethanol in Tris·HCl buffer
(pH 8.5–9.0) for deprotection in water
and ii) by treatment with b-mercapto-
ganic medium. Successful solid-phase
synthesis of hexapeptides Ac-Lys-Asp-
Glu-Val-Asp-Lys
A
has
clearly demonstrated the full orthogon-
ality of these new amino protecting
groups with Fmoc and Boc protections.
The utility of the Ardec orthogonal de-
protection strategy for site-specific
chemical modification of peptides bear-
ing several amino groups was illustrat-
ed firstly by the preparation of a fluo-
rogenic substrate for caspase-3 pro-
tease containing the cyanine dyes
Cy3.0 and Cy5.0 as FRET donor/ac-
ceptor pair, and by solid-phase synthe-
sis of an hexapeptide bearing a single
biotin reporter group.
ethanol
and
1,8-diazobicyclo-
AHCTREUNG
pyrrolidinone for deprotection in an or-
A
Keywords: enzymes · FRET (fluo-
rescence resonance energy transfer) ·
fluorescent probes · peptides · protect-
ing groups · solid-phase synthesis
cal peptide couplings followed by
standard deprotection protocols. They
were used to optimize the conditions
for complete thiolytic removal of the
[e] Dr. G. Turcatti
[a] M. Lapeyre, Prof. P.-Y. Renard, Dr. A. Romieu
IRCOF/LHO, Equipe de Chimie Bio-Organique
UMR 6014 CNRS, INSA de Rouen et UniversitØ de Rouen
1, rue Tesni›res, 76131 Mont-Saint-Aignan Cedex (France)
Fax : (+33)2-35-52-29-59
Manteia Predictive Medicine SA, Zone Industrielle
1267 Coinsins (Switzerland)
[**] Applications of Ardec chemistry to oligonucleotide and peptide syn-
thesis were first described in a patent entitled “Trace-less cleavable
cross-linker reagents and use thereof”, filled on February 12, 2004 at
the European Patent Office: A. Romieu, G. Turcatti, EP 04100542.2.
This patent is based on preliminary experiments done at Manteia Pre-
dictive Medicine SA, a Swiss-based private company (spin-out from
Serono) developing an ultra high-throughput DNA sequencing tech-
nology. This technology was jointly acquired by Lynx Therapeutics
Inc. (www.lynxgen.com) and Solexa Ltd. (www.solexa.com) in March
2004. Both companies completed a merger in March 2005 and became
Solexa Inc.
E-mail: anthony.romieu@univ-rouen.fr
[b] Dr. H. Oulyadi
IRCOF/LCOBS, Laboratoire de RØsonance MagnØtique NuclØaire
UMR 6014 CNRS, INSA de Rouen et UniversitØ de Rouen
1, rue Tesni›res
76131 Mont-Saint-Aignan Cedex (France)
[c] Dr. J. Leprince, Dr. H. Vaudry
INSERM U 413
Laboratoire de Neuroendocrinologie Cellulaire et MolØculaire
IFRMP 23, Place E. Blondel
76821 Mont-Saint-Aignan Cedex (France)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
[d] Dr. M. Massonneau
QUIDD, 24bis, rue Jacques Boutrolle d’Estaimbuc
76132 Mont-Saint-Aignan Cedex (France)
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3655

Introduction
group of nucleotides (e.g., 3’-O-(2-N-dansylethyldithiometh-
yl) derivative of 5’-triphosphate of thymidine 4) to get rever-
sible fluorescent terminators suitable for DNA sequencing
by synthesis.[16] However, the multi-step synthetic procedure
required for the transformation of alcohols to their corre-
sponding alkyldithiomethyl ethers is not suitable for a rou-
tine use of such protecting groups (namely: Pummerer reac-
tion to generate the methylthiomethyl ether, halogenation
and subsequent nucleophilic substitution to generate the
corresponding alkyl- or arylthiosulfonate derivative and fi-
nally a disufide exchange reaction with an alkyl- or an ar-
ylthiol). Extension of this protection strategy to a wide
range of polyfunctional (bio)molecules thus appears tricky.
With the goal in mind to develop a new cleavable disulfide-
based protecting group applied to the biological chemistry
of peptides meeting both the requirement of an easy and
straightforward introduction onto the peptide side chains
and a specific and mild thiolytic deprotection, we have ex-
amined the chemistry of the aryldithioethyloxycarbonyl
functionality. Indeed, we thought that the cleavage of the di-
sulfide bond of 5 with thiols (or other reducing agents)
should generate the unstable 2-thioethyl carbonate (or car-
bamate) 6 which might decompose spontaneously into the
corresponding alcohol (or amine), carbon dioxide and ethyl-
ene episulfide. This kind of intramolecular elimination reac-
tion has already been reported in the literature, especially
for the removal of various S-acyl and disulfide derivatives of
the 2-thioethyl moiety from phosphate esters.[17]
Selective temporary protection or inactivation of a chemi-
cally reactive functionality in biological compounds is an im-
portant tool in the field of organic and bioorganic chemistry
especially for the controlled synthesis of biopolymers (i.e.,
oligonucleotides, oligosaccharides, peptides, and conjugates
thereof).[1–6] The majority of the known protecting groups
are acid or base labile; the conditions for their removal are
often too harsh and can be incompatible with complex bio-
molecules. Consequently, numerous sophisticated protecting
groups have been developed which involve mild deblocking
reagents, for instance the use of enzymes, fluoride ion sour-
ces, noble metals or UV light.[7–10] Surprisingly, few efforts
have been devoted to the design of disulfide bridge contain-
ing protecting groups although they can undergo highly se-
lective reductive cleavage by mild treatment with a thiol or
a trialkylphosphine in various organic solvents or weakly
basic aqueous buffers.[11] The dithiasuccinoyl group 1 has
been developed as an amino protecting group for solid-
phase synthesis of modified peptides and protected peptide
nucleic acids (Scheme 1).[12,13] This group is stable under
strongly acidic conditions and photolysis but it is rapidly and
specifically removed under mild conditions by thiolysis.
Until the recent publication of Barany and Merrifield about
the efficient synthesis of 1,2,4-dithiazolidine-3,5-diones,[14]
the lack of a simple and robust method to prepare Dts-
amines in high yields prevented the widespread use of this
heterocycle as an orthogonal amino protecting group.
Kwiatkowski suggested the protection of hydroxyl groups of
biological compounds with an alkyldithiomethyl moiety 2.[15]
The aminoethyl derivative 3 was used as a 3’-OH blocking
Herein we describe the development of the phenyldithio-
ethyloxycarbonyl and the 2-pyridyldithioethyloxycarbonyl
protecting groups 7 and 8. The novel thiol-labile urethanes
were introduced as lysine side chain protecting groups both
in solution- and solid-phase
peptide synthesis, and used for
the preparation of atypical
peptides.
Results and Discussion
Preparation of Na-Ardec-pro-
tected lysine monomers: Or-
thogonal protection of the e-
amino group of l-lysine with
the Ardec groups firstly re-
quires deactivation of the a-
amino and a-carboxy groups
by formation of a copper(ii)
complex and subsequent N-
alkoxycarbonylation with an
active carbonate derived from
the corresponding (2-aryldi-
thio)ethanol. Chemical meth-
ods for the introduction of var-
ious alkoxycarbonyl moieties
as amino protecting groups are
Scheme 1. Disulfide bridge containing protecting groups already reported in literature and principle of aryldi-
thioethyloxycarbonyl protecting groups.
well documented.[18] To avoid
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Chem. Eur. J. 2006, 12, 3655 – 3671

Amine Protecting Groups
FULL PAPER
the use of unstable and toxic haloformates, several alkoxy-
carbonylating reagents having either heterocyclic (e.g., imi-
dazolyl, N-hydroxysuccinimidyl, 1-hydroxybenzotriazolyl) or
electron-withdrawing group substituted phenol (e.g., 4-nitro-
phenyl, pentafluorophenyl) leaving groups have been devel-
oped. In a first approach, we used the succinimidyl carbo-
nates 13 and 14 (Scheme 2a). Active carbonate 14 was easily
ence of pyridine gave 11 and 12 in 45 and 49% yield, re-
spectively.[20] As previously reported for some unsymmetri-
cal disulfides, 11 was found to be susceptible to dispropor-
tionation to the corresponding symmetrical disulfides 9 and
17 (Scheme 3) both in pure form and in solution in various
organic solvents.[21] This disproportionation reaction was not
Scheme 3. Chemical structure of disulfide 17 and 2-pyridinethiol/2-pyridi-
nethione tautomeric equilibrium.
observed with (2-(2’-pyridyl)dithio)ethanol (12) because the
2-pyridinethiol released is in equilibrium with the 2-pyridi-
nethione which is not able to react with the unsymmetrical
disulfide (Scheme 3).[22] Consequently, (2-phenyldithio)etha-
nol (11) had to be used immediately after its purification by
silica gel chromatography. Treatment of 11 and 12 with 4-ni-
trophenyl chloroformate in dry acetonitrile in the presence
of triethylamine enabled their clean conversion into the N-
alkoxycarbonylation reagents 15 and 16. These activated car-
bonates are stable compounds which were easily purified by
silica gel chromatography. Compound 15 was obtained in a
pure form as a white powder whereas 16 was found to be
contaminated with small amounts (~5%) of 4NP. Interest-
ingly, these reagents could be stored at 48C for several
months without significant degradation.
Selective Ne-acylation of the copper(ii) complex of l-
lysine 18 with Ardec-ONp 15 or 16 was achieved by using
the method reported by Rosowsky and Wright for the intro-
duction of the Teoc protecting group (Scheme 4).[23] Accord-
ingly, removal of the metal ion with ethylenediamine tetra-
acetic acid disodium salt gave the targeted Ne-Ardec-pro-
tected lysine monomers 19 and 20 which were isolated in
good yields by simple filtration; they were obtained in over
Scheme 2. Structure and synthesis of the activated reagents suitable for
the Ardec protecting groups introduction. a) Succinimidyl carbonates,
only 14 was prepared. b) 4-Nitrophenyl carbonates (Ardec-ONp).
prepared from (2-(2’-pyridyl)dithio)ethanol 12 and DSC.
This compound was found to be very stable especially in
aqueous buffers but showed poor acylating reactivity to-
wards amino groups of amino
acids and nucleosides. Conse-
quently, we turned towards the
synthesis of active carbonates
15 and 16 derived from 4-nitro-
phenol (4NP).[19] These inter-
mediates were found to be
more reactive than the corre-
sponding
N-hydroxysuccini-
mide (NHS) derivatives. The
reagents, (2-phenyldithio)ethyl
4-nitrophenyl carbonate (15)
and (2-(2’-pyridyl)dithio)ethyl
4-nitrophenyl carbonate (16),
were prepared from the com-
mercially available diaryl disul-
fide 9 and 10 in two steps
(Scheme 2b). Disulfide ex-
change between b-mercapto-
ethanol and diaryl disulfide 9
or 10 in methanol in the pres-
Scheme 4. Introduction of the Ardec protecting groups onto the e-amino group of lysine.
Chem. Eur. J. 2006, 12, 3655 – 3671
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3657

A. Romieu et al.
95% purity (checked by RP-HPLC) and so they did not re-
quire further purification. Their structures were confirmed
by detailed measurements, including ESI mass spectrometry
and NMR analyses.
Stability studies of the Ardec protecting groups under the
cleavage conditions used for other protecting groups: To
assess the orthogonality and compatibility between Ardec
and other protecting groups currently used in peptide chem-
istry (e.g., allyl, Aloc, Boc, Dde, Fmoc, silyl and photo-
labile protecting groups), stability studies were performed in
Preparation of Na-Fmoc-Ne-Ardec-lysine building blocks
and their use in solution-phase peptide synthesis: The Ne-
solution assays with dipeptides H-Lys(Ardec)-Phe-OH 25
G
Ardec-protected monomers H-Lys
A
and 26. It was found that Ardec groups could be readily
cleaved from the lysine side chain only under aqueous basic
conditions (Table 1, entries 4–5 and 10–11), returning the
Lys(Pydec)-OH (20) were converted into the corresponding
ACHTREUNG
Na-Fmoc building blocks 21 and 22 by using N-hydroxysuc-
cinimidyl 9-fluorenylmethyl carbonate under the standard
conditions reported by Lapatsanis et al. (Scheme 5).[24] The
compounds were isolated by silica gel chromatography and
characterized on the basis of elemental and MS analyses
Table 1. Stability of the Ardec protecting groups to a variety of condi-
tions.[a]
Cleavage conditions (equiv)
Phdec
Pydec
1
and consistent IR and H and 13C NMR spectral data.
1
2
3
4
5
95% TFA/H2O (215)
10% HCl/H2O (3600)
1m Et2NH/CH2Cl2 (30)
2m LiOH/H2O (2200)
2m LiOH/H2O (10)
stable
stable
stable
cleaved
cleaved
stable
stable
stable
To evaluate the suitability of the Phdec and Pydec groups
for peptide chemistry, dipeptides H-Lys
and 26 were synthesized by coupling either Fmoc-Lys-
(Phdec)-OH (21) or Fmoc-Lys(Pydec)-OH (22) with H-Phe-
cleaved
partially cleaved
(21%)
A
ACHTREUNG
OtBu and subsequent removal of the Fmoc and tBu protect-
ing groups. Coupling reaction was achieved with BOP re-
agent in the presence of DIEA in dry dichloromethane.[25]
6
7
8
5% DBU/NMP (180)
5% N2H4/NMP (190)
5% N2H4/NMP (10)
stable
stable
stable
stable
cleaved
partially cleaved
(20%)
The fully-protected dipeptides Fmoc-Lys(Ardec)-Phe-OtBu
A
9
[Pd
G
stable
stable
23 and 24 were isolated by silica gel chromatography in 79
and 97% yields. Thereafter, sequential treatment of 23 and
24 with TFA/H2O (95:5) and diethylamine provided the tar-
geted dipeptides 25 and 26 which were purified by RP-
HPLC. Their structures were confirmed by detailed meas-
urements, including MALDI-TOF mass spectrometry and
NMR analyses. Furthermore, RP-HPLC analyses of the
crude reaction mixtures at each step of this peptide assem-
bly process have clearly shown that no cleavage modifica-
10
11
12
13
1m TBAF in THF (185)[b]
1m TBAF in THF (10)[b]
TEA·3HF (25)
cleaved
cleaved
stable
cleaved
cleaved
stable
UV light (350 nm)/NMP
stable
stable
[a] Stability of the Ardec protecting groups was evaluated by solution
assays with dipeptides H-Lys(Ardec)-Phe-OH 25 and 26. The crude reac-
AHCTREUNG
tion mixtures were analyzed by RP-HPLC (after 1 h of incubation at
room temperature, system E) to detect and quantify the recovered
Ardec-protected dipeptide 25 or 26 and unprotected dipeptide 28 (for
more details, see Experimental Section). [b] Presence of water (solution
of TBAF in THF is highly hygroscopic) may responsible of the cleavage.
tion of Lys(Ardec) residues occurred. Indeed, no trace of di-
A
peptides Fmoc-Lys-Phe-OtBu, Fmoc-Lys-Phe-OH and H-
Lys-Phe-OH were detected after the coupling, tBu and
Fmoc removal steps, respectively. Thus, the Phdec and
Pydec protecting groups are completely stable under usual
conditions applied in peptide synthesis.
unprotected dipeptide H-Lys-Phe-OH (28). The Ardec moi-
eties were found to be stable to all of the other conditions
investigated, but RP-HPLC analysis showed that the reac-
tion with a large excess of hydrazine caused removal of
Scheme 5. Preparation of Na-Fmoc-Ne-Ardec-lysine building blocks and their use in the synthesis of model dipeptides H-Lys
(Ardec)-Phe-OH.
G
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Amine Protecting Groups
FULL PAPER
a)
Pydec (entry 7). Thus, deprotection conditions for the Dde
(or its hindered variant ivDde) amine protecting group
should be carefully checked (i.e., equivalents N2H4) to be
completely orthogonal to Ardec.
100
90
80
70
60
50
40
30
20
10
0
28
DTT 40 equiv
}
27
28
DTT 80 equiv
}
Selective removal of Ardec protecting groups in solution:
We postulated that the Ardec groups are removed in reduc-
tive conditions by thiols or trialkylphosphines through a 2-
thioethyl carbamate intermediate, which spontaneously de-
composes to give the free amino function (Scheme 1). The
reaction is driven to completion by loss of one equivalent of
ethylene episulfide and one equivalent of gaseous carbon di-
oxide. Furthermore, an important aspect of this deprotection
strategy must be the ability to efficiently remove the Ardec
group under mild reducing conditions and in aqueous media
compatible with the stability of peptides (or proteins). To
confirm the mechanism and to define an optimized protocol
for quantitative thiolytic removal of the Ardec groups at the
peptide level, deprotection efficiency was evaluated by solu-
27
7.5
8
8.5
9
pH of Tris.HCl buffer
b)
100
90
80
70
60
50
40
30
20
10
0
28
-Me 40 equiv
β
}
27
28
-Me 80 equiv
β
}
27
tion assays with dipeptides H-Lys(Ardec)-Phe-OH 25 and
A
7.5
8
8.5
9
26.
pH of Tris.HCl buffer
Dipeptide H-Lys(Pydec)-Phe-OH (26) was treated with
A
increasing amounts (i.e., 5, 10, 20, 40 and 80 equiv) of DTT
or b-mercaptoethanol in Tris·HCl buffer, at four different
pH values (i.e., 7.5, 8.0, 8.5 and 9.0) for 30 min. The crude
deprotection reactions were analyzed by RP-HPLC to
detect and quantify the recovered Pydec-protected dipeptide
26, the 2-thioethyl carbamate intermediate 27 and dipeptide
H-Lys-Phe-OH (28) in order to evaluate the rates of disul-
fide reduction and intramolecular elimination. As expected,
the clean and quantitative conversion of 26 into the unpro-
tected dipeptide 28 occurred either using DTT or b-mercap-
toethanol (Figure 1).[26] The identification of H-Lys-Phe-OH
(tR =12.8 min) was confirmed by co-injection with a stand-
O
H2N
H
OH
N
H
S
O
N
O
S
N
O
26
O
H2N
OH
N
H
H
O
N
O
HS
O
27
ard independently prepared from Fmoc-Lys(Boc)-OH and
A
H-Phe-OtBu. When Tris·HCl buffer at pH 7.5 was used, the
quantitative yield of the disulfide reduction step was not af-
fected but the major product from deprotection was the 2-
thioethyl carbamate intermediate 27 (tR =18.5 min). These
results clearly show that a pH value over 8.0 is required for
the decomposition of 2-thioethyl carbamate derivatives This
suggest that the fragmentation reaction of 27 which initially
involves an internal nucleophilic attack of the thiol group on
the a carbon atom of the 2-thioethyl moiety is the key step
of this reductive deprotection mechanism and may be fav-
oured by the formation of a thiolate anion, a better nucleo-
phile than the corresponding thiol. As the pKa value of the
thiol group of 27 is close to 9.0,[27] the limiting step for the
reaction is this acid–base equilibrium leading to the forma-
tion of thiolate anion intermediate. Similar results were ob-
O
H2N
OH
N
H
H2N
O
28
Figure 1. Rates of reductive deprotection of H-Lys(Pydec)-Phe-OH (26)
G
in Tris·HCl buffer as a function of pH. a) reductive deprotection mediat-
ed by DTT. b) Reductive deprotection mediated by b-mercaptoethanol.
Ardec removal was performed with reducing agent/Tris·HCl buffer
(25 mm), 30 min at room temperature. Under these conditions, starting
dipeptide H-Lys(Pydec)-Phe-OH (26) was never detected. Percentages of
27 and 28 were determined by RP-HPLC as reported in Supporting In-
A
formation.
erogeneity of the deprotection mixture (a suspension of im-
miscible PBu3 was observed) despite the use of propan-1-ol
as organic cosolvent.
This reductive deprotection was also investigated in or-
ganic media. Thus, dipeptide H-Lys(Pydec)-Phe-OH (26)
was treated with increasing amounts (i.e., 10, 20, 40 and
80 equiv) of b-mercaptoethanol in NMP for 15 min in the
absence or presence of a base such as DIEA, piperidine,
tained with dipeptide H-Lys(Phdec)-Phe-OH (25) which
A
suggests that structural features of the aryl disulfide bridge
have little influence on the rate of Ardec removal. The use
of tri-n-butylphosphine as the deblocking reagent was also
explored but the deprotection rates were found to be signifi-
cantly lower than with DTT and b-mercaptoethanol (data
not shown). A possible explanation can be found in the het-
N
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3659

A. Romieu et al.
triethylamine and DBU. Preliminary experiments have
shown that the thiolytic removal of the Ardec amino pro-
tecting groups works with a wide range of organic solvents
(i.e., CH2Cl2, CH3CN, DMF, NMP and pyridine). We have
chosen NMP in order to directly transfer these reducing
conditions to solid-phase deprotection. HPLC analyses of
the crude deprotection reactions and subsequent quantifica-
tion of 2-thioethyl carbamate intermediate 27 and dipeptide
H-Lys-Phe-OH (28) have clearly shown that only the addi-
tion of DBU resulted in complete thiolytic removal of the
Pydec group. Indeed, in the absence of base or with piperi-
dine, triethylamine or DIEA, only the 2-thioethyl carbamate
intermediate 27 was detected. The unexpected inefficacy of
secondary and tertiary amines to induce decomposition of
27 into ethylene episulfide and CO2 is in agreement with a
higher pKa value of thiol group in polar aprotic solvents
than in water. The reaction time and amount of DBU were
varied in order to optimize the deprotection conditions.
Quantitative removal of the Pydec group from the lysine
side chain was found to be: b-mercaptoethanol (80 equiv)/
DBU (160 equiv) in NMP for 15 min. When submitted to
the same anhydrous reducing conditions, dipeptide H-Lys-
Indeed, double-labeled peptides are currently used as fluo-
rescent probes based on the principle of fluorescence reso-
nance energy transfer to detect various enzyme activi-
ties.[40,41]
Starting from a Fmoc Rink amide MBHA resin, peptide
chain assembly was carried out by using standard Fmoc/tBu
chemistry (Scheme 6).[42] All amino acids were activated by
(Phdec)-Phe-OH (25) gave similar deprotection rates.
Na-Fmoc-Ne-Ardec-lysine building blocks in solid-phase
peptide synthesis: To check the full orthogonality of the
Ardec groups with Fmoc and Boc protection in solid-phase
peptide synthesis and to demonstrate its utility in the prepa-
ration of highly functionalized peptides, a model hexapep-
tide bearing two lysine residues substituted with two differ-
ent chemical modifications was prepared by using either
Fmoc-Lys
G
ACHTREUNG
Scheme 6. Solid-phase synthesis of peptides(Ardec) 29, 30; [a] overall
N
Hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys
A
yields from Rink amide MBHA resin.
and 30 contain the Asp-Glu-Val-Asp motif which is a specif-
ic substrate for caspase-3.[28] The cleavage site is located
after the aspartic acid residue from the C-terminal side. Cas-
pases belong to the aspartate-specific cysteinyl proteases
that play a critical role as mediators of apoptotic cell
death;[29] in particular caspase-3 has been identified as being
a key mediator of apoptosis of mammalian cells.[30]
Activation of caspase-3 indicates that the apoptotic path-
way has progressed to an irreversible stage and for this
reason there is a growing interest both in identifying caspase
inhibitors to minimize cell death in pathological conditions
and for inducing caspase activation in cancer cells.[31] In ad-
dition, caspase-3 is widely used for monitoring apoptosis in-
duction for general cytotoxicity screening. This interest for
caspase-3 resulted in the development of several assays
using a variety of formats and amenable to high-throughput
screening.[32–37] Peptides 29 and 30 have been chosen in
order to study the selective chemical modification of each
one of the lysine Ne-side-chain amines with two different
fluorescent markers. This will lead to the development of a
novel strategy for the fluorescent labeling of peptides[38] and
the preparation of a novel fluorogenic substrate useful for
detecting apoptosis in whole cells and for cell-based high-
throughput screening of apoptosis inhibitors or inducers.[39]
using HBTU or TBTU/HOBt/DIEA in NMP.[25] The con-
ductimetric analyses of the Fmoc-deprotection steps have
revealed that the coupling of Fmoc-Lys
A
and Fmoc-Lys(Pydec)-OH (22) proceeded with the same ef-
AHCTREUNG
ficiency as the standard Fmoc-protected amino acids. Final
acetylation of the N-terminal side was achieved by treat-
ment with Ac2O/HOBt/DIEA in NMP. The hexapeptides
were simultaneously cleaved from the resin and side chain
deprotected by treatment with TFA in the presence of
phenol, anisole and water. The crude Ardec-protected pepti-
des were isolated by precipitation in cold tert-butyl methyl
ether and analyzed by RP-HPLC and MALDI-TOF mass
spectrometry.
Ac-Lys-Asp-Glu-Val-Asp-Lys
A
(29) and Ac-Lys-Asp-Glu-Val-Asp-Lys
AHCTREUNG
were identified as the major products of the SPPS. However,
a common by-product was observed on the RP-HPLC elu-
tion profiles of the deprotection mixtures and accounted for
25 and 5% of the crude peptide, respectively. The material
was isolated and analyzed by MALDI-TOF mass spectrome-
try from which the homodimer structure 31 was proposed
(m/z 1753.92 [M+H]+, calcd for: 1752.75). Formation of 31
may be explained by a disproportionation reaction of the di-
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Amine Protecting Groups
FULL PAPER
sulfide bridges of two Ardec-protected peptide resin bound
lective labeling of peptides bearing several amino groups
was illustrated by the preparation of 34, a FRET substrate
of caspase-3 protease (Scheme 8). The hexapeptide Ac-Lys-
Asp-Glu-Val-Asp-Lys-NH2 was doubly modified with cya-
nine dyes Cy3.0 and Cy5.0 which proves their utility in a
wide range of bioanalytical applications due to their high ex-
tinction coefficients, high fluorescence quantum yields and
resistance to photobleaching.[43,44] The fluorescent donor
Cy3.0 dye was coupled to the side chain of the free lysine of
units, with subsequent release of
a diaryl disulfide
(Scheme 7). The tendency of homodimerization is probably
Ac-Lys-Asp-Glu-Val-Asp-Lys(Pydec)-NH2 (30) using the
G
corresponding N-hydroxysuccinimidyl ester in dry NMP in
the presence of DIEA. Purification by RP-HPLC provided
the Cy3.0 labeled peptide 32 in 60% yield. The clean re-
moval of the Pydec protecting group from the lysine residue
of 32 was achieved by treatment with an excess of DTT
(50 equiv) in Tris·HCl buffer (pH 9.0). The resulting unpro-
tected hexapeptide 33 was isolated by RP-HPLC in quanti-
tative yield. Finally, the fluorescent acceptor Cy5.0 dye was
attached to the free amino group of 33 by treatment with
the corresponding N-hydroxysuccinimidyl ester in a mixture
of sodium bicarbonate buffer (pH 8.5) and NMP. The fluo-
rogenic caspase-3 substrate was isolated by RP-HPLC in a
moderate 30% yield and its structure was confirmed by
MALDI-TOF mass spectrometry.[45] When we measured the
fluorescence spectrum of this probe after excitation of the
donor Cy3.0 at 540 nm, a major emission at 665 nm corre-
sponding to the Cy5.0 fluorescence as well as a minor emis-
sion at 562 nm corresponding to the remaining untransferred
Cy3.0 fluorescence were observed (Figure 2). The emission
Scheme 7. Proposed mechanism for the formation of peptide dimer 31.
favoured by the high effective concentration of reactive sites
at the resin surface (resin loading) because this type of di-
sulfide homodimer was not detected during the solution-
phase synthesis of dipeptides 25 and 26. However, this side
reaction significantly occurs only during the synthesis of
Phdec-protected peptides because only the unsymmetrical
phenyl disulfide derivatives are prone to disproportionation
to the corresponding symmetrical disulfides. Further experi-
ments are in progress in order to improve the solid-phase
synthesis, as use of a resin of a lower loading. RP-HPLC pu-
Figure 2. Fluorescence emission spectra (excitation at 540 nm) of pepti-
des 33 labeled with Cy3.0 only (c) and 34 labeled with Cy3.0 and
Cy5.0 (a) in HPLC grade water at 258C, concentration 0.53 mm.
rification of Ac-Lys-Asp-Glu-Val-Asp-Lys
A
of Cy5.0 (acceptor) resulting from the excitation of Cy3.0
(donor) occurred via the non-radiative transfer from the
donor to the acceptor according to the theory of Fçrster.[46]
The efficiency of this energy transfer (E) is typically meas-
ured using the relative fluorescence intensity of the donor
(Cy3.0), in the absence (FD) and presence (FDA) of acceptor
and Ac-Lys-Asp-Glu-Val-Asp-Lys(Pydec)-NH2 (30) provid-
AHCTREUNG
ed the pure products (purity > 95%) in overall yields from
Fmoc Rink amide MBHA resin of 17 and 15%, respectively.
They displayed the expected molecular weights of the
Ardec-protected peptides m/z 986.73 ([M+H]+, calcd for:
985.38) and m/z 987.52 ([M+H]+, calcd for: 986.38), as indi-
cated by MALDI-TOF mass spectrometry analyses.
(Cy5.0): E = 1ꢀ(FDA/FD). For our fluorescent probe, this
A
was accomplished by comparing the Cy3.0 emission for pep-
tides 33 and 34. As depicted in Figure 2, the value of FDA/FD
is close to 0.10 so that the transfer efficiency is approximate-
ly 90% (E=0.89). The FRET efficiency E depends on the
Fluorescent labeling of Ardec-protected peptides: The utili-
ty of the Ardec orthogonal deprotection strategy for the se-
Chem. Eur. J. 2006, 12, 3655 – 3671
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3661

A. Romieu et al.
a)
inverse-sixth-power of the distance between the donor and
acceptor: E=1/[1 +
(R/R0)6] where R0 is the distance at
600
400
200
0
A
ACHTREUNG
which half of the energy is transferred (i.e., Fçrster dis-
tance), and depends on the spectral characteristics of the
dyes and their relative dipole orientation.[47] Thus, the
FRET results allow to estimate the distance between the
two cyanine dye molecules within peptide 34. If one assumes
an R0 value of 53 Š for the Cy3.0/Cy5.0 FRET pair (previ-
ously determined by Ishii et al.)[48] and a random dipole–
dipole orientation value of 2/3 for the orientation factor k2,
a distance of 37ꢁ1.0 Š is obtained. In general, FRET is
better suited for detecting changes in distances rather than
absolute distances because of the dependence on the rela-
tive orientation of the dyes, the k2 factor, which is often
poorly understood. The efficiency of the energy transfer
(donor quenching) obtained with the doubly labeled sub-
strate 34 is high enough to use this read-out for monitoring
caspase-3 activity. Enzymatic action will result from de-
quenching of donor fluorescence upon separation of the two
dyes as a consequence of enzymatic peptide bond cleavage.
As expected, incubation of peptide 34 with recombinant
human caspase-3 has resulted in an appreciable increase of
the fluorescence emission of Cy3.0 (Figure 3a,b). This de-
quenching effect of the donor is concomitant with a de-
crease of Cy5.0 acceptor emission due to the cancellation of
FRET resulting from the protease cleavage. The donor fluo-
rescence change is in agreement with an approximate effi-
ciency of transfer of 90% suggesting that the peptide bond
cleavage reaction induced by caspase-3 went to completion.
Direct excitation of the accep-
550
600
650
700
750
Wavelength [nm]
b)
Em. λ = 565 nm
Em. λ = 667 nm
600
400
200
2000
4000
6000
t [s]
8000
10000
Figure 3. a) Fluorescence emission spectra (excitation at 540 nm) of pep-
tide 34 in caspase-3 buffer at 258C (concentration 0.53 mm) with (c) or
without (a) incubation of recombinant human caspase-3 (0.008 U, in-
cubation time 3 h). b) Fluorescence emission time course (excitation at
540 nm) of peptide 34 with recombinant human caspase-3 at 565 and
667 nm.
groups on the solid-phase, estimated on the basis of the re-
sults of solution deprotection assays, were b-mercaptoetha-
nol (0.5m)/DBU (1.0m) in NMP for 30 min. Under these re-
tor at 640 nm also resulted in a
significant increase of the fluo-
rescence emission. Further-
more,
a control reaction in
which peptide 34 was incubat-
ed only with the caspase-3
buffer, did not give non-specif-
ic cleavage of this probe.[45]
Selective removal of Ardec
protecting groups on solid-
phase: As a further proof for
the compatibility of the Ardec
groups with Fmoc/tBu strategy,
solid-supported deprotection
and subsequent chemical solid-
supported derivatization of a
distinct lysine residue within
the protected hexapeptide
resin Ac-Lys
Glu(OtBu)-Val-Asp
Lys(Phdec)-Rink
A
(OtBu)-
A
ACHTREUNG
ACHTREUNG
MBHA R1 was performed
(Scheme 9).
Suitable
conditions
for
quantitative removal of the
Ardec-amino
3662
protecting
Scheme 8. Preparation of the energy-transfer substrate of caspase-3 enzyme 34.
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3655 – 3671

Amine Protecting Groups
FULL PAPER
remain intact. The selective de-
protection of the Phdec group
was followed by acylation of the
resulting amino group with a
biotinylation reagent (EZ-Link
NHS-LC-biotin) in dry NMP in
the presence of DIEA. Depro-
tection of all the other side
chains and simultaneous cleav-
age from the resin yielded, after
precipitation in cold TBME, the
crude biotinylated peptide 35
which was finally purified by
RP-HPLC (yield: 40%). For this
latter chromatographic purifica-
tion, it was essential to use a
slow linear gradient of CH3CN
(0.25%minꢀ1) in aqueous TFA
to remove a side product which
accounted for 10% of the crude
biotinylated peptide and identi-
fied as an aspartimide derivative
36a or 36b (Scheme 9b, m/z
1095.65 [M+H]+, calcd for:
1094.54). The aspartimide for-
mation is one the best docu-
mented side reactions in peptide
synthesis.[49] This sequence-de-
pendent cyclization is catalyzed
both by acids and bases and fre-
quently occurs during the solid-
phase synthesis of peptides bear-
ing the Asp–Gly motif. Further-
more, it was shown that the use
of harsh Fmoc cleavage condi-
tions (i.e., use of DBU instead
of piperidine) promoted the as-
partimide formation even for
Asp–AA motifs where Gly is re-
placed by more sterically hin-
dered amino acids (e.g., Ala,
His
N
N
is likely that our reductive de-
protection conditions partly af-
fected the integrity of the Asp-
A
N
A
ACHTREUNG
suggested by Mergler et al.,[49b]
this side reaction could have
been suppressed by incorpora-
tion of Asp residues bearing a b-
carboxylic acid protecting group
bulkier than tBu ester (3-methyl-
pent-3-yl ester). Analysis of the
Scheme 9. a) Solid-phase synthesis of biotinylated peptide 35. b) Structure of aspartimide side products 36a
and 36b; [a] overall yield from Rink amide MBHA resin.
ductive deprotection conditions, the peptide remains still
bound to the resin and all acid-labile protecting groups (in-
cluding the Boc of lysine residue from the N-terminal side)
conjugate 35 using MALDI-TOF mass spectrometry indicat-
ed that the biotin derivative was attached only once to the
peptide (Figure 4).
Chem. Eur. J. 2006, 12, 3655 – 3671
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3663

A. Romieu et al.
1113.6781
([MM+H])
100
90
80
70
60
50
40
30
20
10
0
2.7E+4
+
Ac-Lys-Asp-Glu-Val-Asp-Lys(Ahx-Biotin)NH2 (35)
1115.6812
1135.6814
1097.6547
1117.6767
1153.6494
1153.6494
600.3789
600.3789
998.6286
998.6286
0
499.0
899.4
1299.8
1700.2
2100.6
2501.0
m / z
Figure 4. MALDI-TOF mass spectrum of the biotinylated peptide 35, in the positive mode, a-cyano-4-hydroxycinnamic acid was used as matrix,
[M+H]+: m/z: calcd for: 1113.56, found 1113.67.
Since removal of the Ardec protecting group was cata-
lyzed by a strong base such as DBU it was necessary to
NxH (ahx)
check if racemization of some amino acids occurred during
NxH (K6)
the deprotection process.[50] HPLC and 1H NMR analyses
D2 K1
D5
E3
V4
K6
were considered suitable for this purpose. On the RP-HPLC
elution profiles of the biotinylated peptide 35 before and
after purification, a single sharp peak was observed reflect-
ing the lack of diastereoisomeric peptide mixture.[45] Fur-
thermore, this was unambiguously confirmed by 1D and 2D
1H NMR spectra which are consistent with the presence of a
single diastereomer (Figure 5). Assignment of the proton
NMR spectrum of biotinylated peptide 35 in pure methanol
at 208C was carried out by the sequential assignment strat-
egy proposed by Wüthrich.[51] The first step involved analy-
δ1H
1.0
1.5
2.0
2.5
3.0
3.5
4.0
1
sis of coupling based two dimensional NMR spectra H,1H
1
COSY and H,1H TOCSY experiments to identify spin sys-
tems characteristic of particular amino acids. Then, the spin
system residues were assigned to specific locations in their
1
sequence by observation in the H,1H NOESY spectrum of
NOE signals between resonances of sequentially adjacent
residues. All resonance assignments are summarized in
Table 2 (see Experimental Section). From these experi-
ments, we concluded that despite the use of a strong base
such as DBU, the Ardec deprotection process did not cause
detectable racemization of amino acids in the biotinylated
peptide 35. This is one of the most important requirements
which must be fulfilled by a new temporary or semiperma-
nent protecting group designed for a routine use in peptide
synthesis.
V4
K6
K1
E3
H2
4.5
D2
D5
Conclusion and Perspectives
8.6
8.4
8.2
8.0
7.8
δ1H
In summary, new disulfide-containing protecting groups for
amines are described. The aryldithioethyloxycarbonyl pro-
tecting groups are easily removed by thiols under mild basic
conditions. They are stable to a wide variety of conditions,
Figure 5. Part of the TOCSY spectrum of biotinylated peptide 35, record-
ed in MeOH at 208C with a spin lock time of 80 ms. In this spectrum, are
shown the connectivities among the amide proton (top) and its aliphatic
protons (side chain protons, left), indicated by the residue number.
3664
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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3655 – 3671

Amine Protecting Groups
FULL PAPER
Table 2. Resonance assignments (spectra recorded in MeOH at 208C).
Experimental Section
Abbreviations: Ahx: aminohexanoic
acid, Aloc: allyloxycarbonyl, Ardec:
aryldithioethyloxycarbonyl,
Ardec-
ONp: aryldithioethyloxycarbonyl 4-
nitrophenyl carbonate, b-Me: b-mer-
captoethanol, BOP: benzotriazol-1-yl-
N-oxytris(dimethylamino)phosphoni-
um hexafluorophosphate, CHAPS: 3-
[(3-cholamidopropyl)dimethylammo-
nio]-1-propanesulfonate, CHCA: a-
cyano-4-hydroxycinnamic acid, Cy3.0
dye: 3H-indolium-1-(5-carboxypen-
tyl)-2-[(1E,3E)-3-(1-ethyl-1,3-dihydro-
3,3-dimethyl-5-sulfo-2H-indol-2-yl-
idene)-1-propenyl]-3,3-dimethyl-5-
sulfo-, inner salt, Cy5.0 dye: 3H-indo-
lium, 2-[(1E,3E,5E)-5-[1-(5-carboxy-
pentyl)-1,3-dihydro-3,3-dimethyl-5-
sulfo-2H-indol-2-ylidene]-1,3-penta-
dienyl]-1-ethyl-3,3-dimethyl-5-sulfo-,
inner salt, DBU: 1,8-diazobicyclo-
AA residue
NH
Ha
Hb
Hg
Hd
He
Others
CH3: 2.00 (Ac)
NH3+: 7.74
Lys (K1)
Asp (D2)
Glu (E3)
Val (V4)
Asp (D5)
Lys (K6)
8.31
8.44
8.28
7.82
8.14
8.01
4.28
4.65
4.32
4.06
4.69
4.25
1.80/1.70
2.93/2.85
2.15/2.01
2.15
2.93
1.74
1.46
1.67
2.95
2.44
0.97
2.80
1.89
1.50/1.38
3.16
NH: 7.94
NH2: 7.40/7.15 (CONH2)
Ahx
NHx
2”Ha
2”Hb
2”Hg
1.33
2”H-6
1.75/1.63
2”Hd
2”He
3.17
8.00
2.17
1.62
1.51
biotin
2”NH
H-2
4.5
2”H-3
2.94/2.7
H-4
4.30
H-5
3.22
2”H-7
1.43
2”H-8
1.60
2”H-9
2.17
AHCTREUNG
[a]
–
[a] Not observed due to exchange with residual water of MeOH.
amine, DSC: N,N’-disuccinimidyl car-
bonate, Dts: dithiasuccinoyl, DTT: di-
thiothreitol,
EZ-Link
NHS-LC-
biotin: N-hydroxysuccinimidyl-6-(bio-
including many that are used for the removal of other pro-
tecting groups (allyl, Aloc, Boc, Dde, Fmoc, silyl and photo-
labile protecting groups). The utility of the Ardec moiety as
protecting group of the lysine side chain is shown with ex-
amples from peptide synthesis in solution and on solid sup-
port. Since the Pydec moiety is not prone to the disulfide
disproportionation reaction leading to the formation of pep-
tide dimers, this protecting group is a more valuable tool
than Phdec especially for the synthesis of large peptides in-
tended for post-synthetic modifications in solution. Further-
more, the use of mild reducing conditions has enabled the
selective deprotection of lysine side chain and its subsequent
chemical modification with molecular tags such as biotin
and cyanine dyes. Thus, the Ardec groups may be a useful
alternative to the well-known Dde (or its hindered variant
ivDde) and Mtt groups which are removed by treatment
with bivalent nucleophiles (hydrazine and hydroxylamine)
and by treatment with TFA (1% in CH2Cl2), respectively,
especially for the preparation of cyclic and branched pepti-
des.[52,53] The extension of the Ardec chemistry to amino
acids bearing an hydroxyl group within their side chains
(serine, threonine, tyrosine) is currently under way and will
be reported in due course. It should allow the synthesis of
fully functional and sensitive post-translationally site-specific
modified proteins (glyco- and lipoproteins) which play im-
portant roles in numerous biological processes.[54] Further-
more, in order to get functionalized biopolymers suitable for
chemoselective ligation reactions,[55,56] the Ardec-based pro-
tection strategy might be extended to other chemical func-
tional groups such as aldehydes and ketones, for which few
protecting groups removable under mild conditions are
available.[57]
tinamido)hexanoate, FRET: fluorescence resonance energy transfer,
HBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate, HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid,
HOBt: N-hydroxybenzotriazole, NHS: N-hydroxysuccinimide, ivDde: 1-
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl, Mpe: 3-meth-
ylpent-3-yl, Mtt: 4-methyltrityl, NHS: N-hydroxysuccinimide, NMM: N-
methylmorpholine, NMP: N-methylpyrrolidinone, 4NP: 4-nitrophenol,
Phdec: phenyldithioethyloxycarbonyl, Pydec: 2-pyridyldithioethyloxycar-
bonyl, TBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tet-
rafluoroborate, TEAA: triethylammonium acetate, Teoc: (2-trimethylsi-
lyl)ethyloxycarbonyl, Tris·HCl: tris(hydroxymethyl)aminomethane hydro-
chloride, TSTU: O-(N-succinimidyl)-N,N,N’,N’-tetramethyluronium tetra-
fluoroborate.
General methods: Column chromatographic purifications were per-
formed on silica gel (40–63 mm) from SdS. TLC were carried out on
Merck DC Kieselgel 60 F-254 aluminium sheets. Compounds were visual-
ized by one or more of the following methods: 1) fluorescence quench-
ing, 2) spray with a 0.2% (w/v) ninhydrin solution in absolute ethanol, 3)
spray with a 3.5% (w/v) phosphomolybdic acid solution in absolute etha-
nol. Acetonitrile was freshly distilled over CaH2 under an argon atmos-
phere prior to use. Dichloromethane was dried by distillation over P2O5.
DIEA and triethylamine were distilled from CaH2 and stored over BaO.
Sulfocyanine dyes Cy3.0 and Cy5.0 were prepared by using literature
procedures.[44] EZ-Link NHS-LC-biotin was purchased from Pierce. Re-
combinant human caspase-3 enzyme (5.52 Umgꢀ1) was purchased from
Sigma. The HPLC grade solvents (CH3CNand MeOH) were obtained
from Acros. Aqueous buffers for HPLC were prepared using water puri-
fied with a Milli-Q system. 1H and 13C NMR spectra were recorded on a
Bruker DPX 300 or AMX-400 spectrometer (Bruker, Wissembourg,
France). Chemical shifts are expressed in parts per million (ppm) from
CD3CN( dH =1.96, dC = 1.32 (CH3), 118.26 (CN)), CD2Cl2 (dH =5.30,
dC =53.52), CDCl3 (dH =7.26, dC =77.36), [D6]DMSO (dH =2.54, dC =
40.45) or D2O (dH =4.79).[58] J values are given in Hz. 13C substitution
was determined with a JMOD pulse sequence, differentiating signals of
methyl and methine carbons pointing “down” (ꢀ) from methylene and
quaternary carbons pointing “up” (+). NMR experiments related to bio-
tinylated peptide 35 were achieved at 208C on a Bruker DMX 600 spec-
trometer (Bruker, Wissembourg, France) equipped with a 5 mm triple
Chem. Eur. J. 2006, 12, 3655 – 3671
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3665

A. Romieu et al.
resonance (1H, 13C, 15N) Cryoprobe including shielded
z
gradients.
from 0 to 60% of CH3CN(40 min)] at a flow rate of 1.0 mLmin ꢀ1. Dual
UV detection was achieved at 260 and 550 nm. System J: RP-HPLC
(Waters XTerra MS C18 column, 5 mm, 7.8”100 mm) with CH3CNand
aq. formic acid as the eluents [100% aq. formic acid (5 min), linear gradi-
ent from 0 to 20% (20 min) and 20 to 50% (60 min) of CH3CN] at a
flow rate of 2.5 mLminꢀ1. UV detection was achieved at 260 nm. System
K: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 4.6”150 mm)
with CH3CNand aq. TFA as eluents [100% aq. TFA (5 min), linear gra-
dient from 0 to 15% (10 min) and 15 to 60% (90 min) of CH3CN] at a
flow rate of 1.0 mLminꢀ1. Triple UV detection was achieved at 260, 550
and 650 nm. System L: RP-HPLC (Waters XTerra MS C18 column, 5 mm,
7.8”100 mm) with CH3CNand aq. TFA as eluents [100% aq. TFA
Bruker XWINNMR software was used to acquire and process the 1D
and 2D spectra. The one and two-dimensional spectra were collected
with carrier frequency in the middle of the spectrum coinciding with the
water resonance which was suppressed by either presaturation using con-
tinuous irradiation during relaxation delay or by using a gradient pulse
Watergate sequence.[59] TOCSY spectra was acquired with a 80 ms spin-
lock time and NOESY experiment was performed using mixing time of
300 ms. 1H chemical shifts were measured relative to the residual signal
of the methyl group of the methanol whose shift relative to tetramethylsi-
lane was arbitrarily chosen at 3.31 ppm. Optical rotations were measured
by using a Perkin Elmer 341 polarimeter. Infrared (IR) spectra were re-
corded as thin-film on sodium chloride plates or KBr pellets using a
Perkin Elmer FT-IR Paragon 500 spectrometer with frequencies given in
reciprocal centimeters (cmꢀ1). UV-visible spectra were obtained on a
Varian Cary 50 scan spectrophotometer. Fluorescence spectroscopic stud-
ies were performed with a Varian Cary Eclipse spectrophotometer. Mass
spectra were obtained with a Micromass Quattro Micro QAA118 spec-
trometer or a Finnigan LCQ-ion trap apparatus equipped with an elec-
trospray source except for the volatile compounds 11 and 12 which were
analyzed with a Thermoquest Finnigan Trace GC coupled to an Auto
Mass Multi III analyzer using electron impact ionization. The purified
peptides were characterized by MALDI-TOF mass spectrometry on a
Voyager DE PRO in the reflector mode with CHCA as a matrix.
(5 min), linear gradient from
0 to 10% (10 min) and 10 to 50%
(160 min) of CH3CN(40 min)] at a flow rate of 2.5 mLmin ꢀ1. UV detec-
tion was achieved at 218 nm.
2-(Phenyldisulfanyl)ethanol (11): Diphenyl disulfide (7.0 g, 31.8 mmol)
was dissolved in a mixture of pyridine and MeOH (300 mL, 1:99). b-Mer-
captoethanol (2.1 mL, 30 mmol) was added dropwise to the stirred solu-
tion and the mixture was left to stir at room temperature overnight. The
reaction was checked for completion by TLC (cyclohexane/ethyl acetate
1:1) and the mixture was evaporated to dryness. The resulting residue
was purified by chromatography on a silica gel column (350 g) with a
step gradient of ethyl acetate (0!50%) in cyclohexane as the mobile
phase, giving 11 as a colorless oil (2.51 g, 13.5 mmol, 45%). Rf =0.62 (cy-
clohexane/ethyl acetate 1:1); 1H NMR (300 MHz, CDCl3): d=1.80 (brs,
Gas chromatography separations: Compounds 11 and 12 were analyzed
with the following chromatographic system: J&W Scientific DB5-MS ca-
pillary column (0.25 mm”30 m) coated with a 0.25 mm film of methylsi-
loxane substituted by 5% phenylsiloxane. The constant pressure was
100 kPa. The injection was performed in the split mode with the tempera-
ture of the injection port set at 2508C. The temperature of the GC oven
was maintained at 508C for 2 min and then raised to 2508C at a rate of
258Cminꢀ1 and finally left at that latter temperature for 35 min.
1H, OH), 2.83 (t, 3J
(H,H)=5.9 Hz, 2H), 3.79 (m, 2H), 7.15–7.20 (m,
R
3H), 7.24–7.29 ppm (m, 2H); 13C NMR (75.5 MHz, CDCl3): d=41.6,
60.2, 127.5, 128.3 (2C), 129.5 (2C), 137.3 ppm; IR (neat): nmax =1021,
1067, 1154, 1218, 1296, 1437, 1474, 1577, 2873, 2924, 3355 cmꢀ1 (broad);
HPLC (system A): tR =24.1 min, purity 96%; UV/Vis (recorded during
the HPLC analysis): lmax =244 nm; GC-MS: tR =12.8 min; m/z: calcd for
+
+
C8H10OS2: 186.30; found: 186.0 [M + ], 142.0 [PhSS ], 110.0 [PhS ]; ele-
mental anlysis calcd (%) for C8H10OS2: C 51.58, H 5.41, S 34.42; found:
C 51.69, H 5.61, S 34.38.
C
C
HPLC separations: Several chromatographic systems were used for the
analytical experiments and the purification steps. System A: RP-HPLC
(Thermo Hypersil GOLD C18 column, 5 mm, 4.6”150 mm) with CH3CN
and triethylammonium acetate buffer (TEAA, 0.1m, pH 7.0) as eluents
2-(Pyridin-2-yldisulfanyl)ethanol (12): Bis(2-pyridyl) disulfide (2.2 g,
10.0 mmol) was dissolved in a mixture of pyridine and MeOH (100 mL,
1:99). b-Mercaptoethanol (0.7 mL, 10 mmol) was added dropwise to the
stirred solution and the mixture was left to stand at room temperature
overnight. The reaction was checked for completion by TLC (CH2Cl2/
ethyl acetate 8:2) and the mixture was evaporated to dryness. The result-
ing residue was purified by chromatography on a silica gel column
(100 g) with a step gradient of ethyl acetate (0!5%) in dichloromethane
as the mobile phase, giving 12 as a colorless oil (0.92 g, 4.9 mmol, 49%).
Rf =0.59 (CH2Cl2/ethyl acetate 8:2); 1H NMR (300 MHz, CDCl3): d=
[100% TEAA (2 min), linear gradient from
0 to 80% of CH3CN
(40 min)] at a flow rate of 1.0 mLminꢀ1. Dual UV detection was achieved
at 254 and 285 nm. System B: RP-HPLC (Thermo Hypersil GOLD C18
column, 5 mm, 4.6”150 mm) with CH3CNand 0.1% aqueous trifluoro-
acetic acid (aq. TFA, 0.1%, v/v, pH 2.0) as eluents [100% aq. TFA
(5 min), linear gradient from 0 to 60% of CH3CN(30 min)] at a flow
rate of 1.0 mLminꢀ1. Triple UV detection was achieved at 210, 260 and
285 nm. System C: RP-HPLC (Thermo Hypersil GOLD C18 column,
5 mm, 4.6”150 mm) with CH3CNand aq. TFA as the eluents [80% aq.
TFA (5 min), linear gradient from 20 to 90% of CH3CN(35 min)] at a
flow rate of 1.0 mLminꢀ1. UV detection was achieved at 254 nm. System
D: RP-HPLC (Interchrom Nucleosil C18 column, 5 mm, 10.0”250) with
2.93 (t, 3J
(H,H)=5.1 Hz, 2H), 3.79 (m, 2H), 5.76 (brs, 1H, OH), 7.12–
N
7.60 (m, 3H), 8.48–8.50 ppm (m, 1H); 13C NMR (75.5 MHz, CDCl3): d=
43.0, 58.5, 121.9, 122.3, 137.2, 150.2, 159.4 ppm; IR (neat): nmax =1045,
1063, 1117, 1147, 1285, 1417, 1455, 1558, 1574, 2865, 2923, 3047,
3332 cmꢀ1 (broad); HPLC (system A): tR =17.8 min, purity 91%; UV/Vis
(recorded during the HPLC analysis): lmax =237 nm, 281 nm; MS (ESI,
CH3CNand aq. TFA as the eluents [100% aq. TFA (5 min), linear gradi-
ent from 0 to 50% of CH3CN(50 min)] at a flow rate of 4.0 mLmin
ꢀ1
.
UV detection was achieved at 260 nm. System E: RP-HPLC (Thermo
Hypersil GOLD C18 column, 5 mm, 4.6”150 mm) with CH3CNand aq.
TFA as the eluents [100% aq. TFA (2 min), linear gradient from 0 to
60% of CH3CN(32 min)] at a flow rate of 1.0 mLmin ꢀ1. Triple UV de-
tection was achieved at 260, 285 and 340 nm. System F: RP-HPLC
(Vydac C18 column, 5 mm, 22.0”250) with CH3CN/TFA (99.9:0.1) and aq.
TFA as the eluents [90% aq. TFA (5 min), linear gradient from 10 to
40% of CH3CN(40 min)] at a flow rate of 10.0 mLmin ꢀ1. Dual UV de-
tection was achieved at 215 and 280 nm. System G: RP-HPLC (Vydac
C18 column, 5 mm, 4.6”250) with CH3CN/TFA (99.9:0.1) and aq. TFA as
eluents [90% aq. TFA (5 min), linear gradient from 10 to 90% of
CH3CN(40 min)] at a flow rate of 1.0 mLmin ꢀ1. Dual UV detection was
achieved at 215 and 280 nm. System H: RP-HPLC (Waters XTerra MS
C18 column, 5 mm, 7.8”100 mm) with CH3CNand aq. formic acid (0.1%,
v/v) as eluents [100% aq. formic acid (5 min), linear gradient from 0 to
20% (20 min) and 20 to 80% (80 min) of CH3CN] at a flow rate of
2.5 mLminꢀ1. UV detection was achieved at 260 nm. System I: RP-
HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 4.6”150 mm) with
CH3CNand aq. TFA as eluents [100% aq. TFA (5 min), linear gradient
positive mode): m/z: calcd for C7H9NOS2: 187.28; found: 187.46 [M+H]+
+
; GC-MS: tR =9.6 min, m/z: 187.0 [M + ], 111.0 [PyS ]; elemental analy-
sis calcd (%) for C7H9NOS2: C 44.89, H 4.84, N7.48, S 34.24; found: C
44.64, H 4.63, N7.88, S 34.21.
C
C
2-(Pyridin-2-yldisulfanyl)ethyl N-hydroxysuccinimidyl carbonate (14): 2-
(Pyridin-2-yldisulfanyl)ethanol (12) (0.38 g, 2.0 mmol) was dissolved in
dry CH3CN(15 mL). Triethylamine (0.55 mL, 4.0 mmol) and DSC
(0.62 g, 2.4 mmol) were added and the reaction mixture was stirred at
room temperature overnight under an argon atmosphere. The reaction
was checked for completion by TLC (CH2Cl2/ethyl acetate 8:2) and the
mixture was evaporated to dryness. The resulting residue was purified by
chromatography on a silica gel column (30 g) with a step gradient of
ethyl acetate (0!5%) in dichloromethane as the mobile phase, giving
activated carbonate 14 as a pale yellow oil (0.39 g, 1.2 mmol, 60%). Rf =
1
0.69 (CH2Cl2/ethyl acetate 8:2); H NMR (400 MHz, CD2Cl2): d=2.74 (s,
4H), 3.08 (t, 3J
C
ACHTREUNG
3666
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3655 – 3671

Amine Protecting Groups
FULL PAPER
151.7, 159.2, 169.0 ppm (2C); UV/Vis (water): lmax (e)=290 (8188), 371
(20702 molꢀ1 dm3 cmꢀ1); HRMS (ESI, positive mode): m/z: calcd for
C12H13N2O5S2: 329.0265, found 329.0269 [M+H]+.
[D6]DMSO + 5% [D]TFA): d=1.34–1.43 (m, 4H), 1.78 (m, 2H), 3.01
3
(m, 4H), 3.93 (m, 1H), 4.18 (t, J
ACHTREUNG
8.30 ppm (brs, 1H, NH3+); 13C NMR (75.5 MHz, [D6]DMSO
+
[D]TFA): d=22.6, 29.8, 30.5, 38.2, 40.5, 52.7, 62.3, 128.2 (3C), 130.3
(2C), 137.3, 156.8, 172.0 ppm; IR (KBr): nmax =687, 736, 1029, 1140, 1240,
2-(Phenyldisulfanyl)ethyl 4-nitrophenyl carbonate (15): 2-(Phenyldisulfa-
nyl)ethanol (11) (2.34 g, 12.6 mmol) was dissolved in dry CH3CN
(35 mL). Triethylamine (2.1 mL, 15.1 mmol) was added and the resulting
mixture was cooled at ꢀ208C. Then, 4-nitrophenylchloroformate (3.0 g,
14.9 mmol) was added and the reaction mixture was stirred at room tem-
perature overnight under N2 atmosphere. The reaction was checked for
completion by TLC (cyclohexane/ethyl acetate 7:3) and the mixture was
evaporated to dryness. The resulting residue was purified by chromatog-
raphy on a silica gel column (200 g) with a step gradient of ethyl acetate
(0!20%) in cyclohexane as the mobile phase, giving activated carbonate
15 as a white powder (2.3 g, 6.5 mmol, 53%). Rf =0.53 (cyclohexane/ethyl
acetate 7:3); m.p. 528C (decomp); 1H NMR (300 MHz, CDCl3): d=3.05
1251, 1273, 1326, 1415, 1534, 1579, 1688, 2868, 2948, 3049, 3342 cmꢀ1
;
HPLC (system B): tR =26.4 min, purity 94%; UV/Vis (recorded during
the HPLC analysis): lmax =234 nm; MS (ESI, positive mode): m/z: calcd
for C15H22N2O4S2: 358.48; found: 359.09 [M+H]+, 716.99 (dimer)
[2M+H]+; elemental analysis calcd (%) for C15H22N2O4S2: C 50.26, H
6.19, N7.81, S 17.89; found: C 49.48, H 6.23, N7.74, S 16.81.
Ne-(2-(Pyridin-2-yldisulfanyl)ethyloxycarbonyl)-l-lysine (20): l-Lysine
monohydrochloride (0.38 g, 2.1 mmol) was dissolved in distilled water
(3 mL) and basic CuCO3 (i.e., CuCO3·Cu(OH)2·H2O, 0.30 g, 1.25 mmol)
was added. The resulting mixture was heated under reflux for 2 h and
then the hot suspension was filtered over Celite 545 and washed with dis-
tilled water (~20 mL). After cooling to 48C, the reaction mixture was ba-
sified to pH 9–10 with Na2CO3 (1.1 g, 10.5 mmol) and activated carbonate
16 (0.62 g, 1.8 mmol) dissolved in CH3CN(12 mL) was added. After stir-
ring at room temperature overnight, a green precipitate was formed. The
reaction mixture was acidified to pH ~7 with glacial acetic acid and
CH3CNwas removed under reduce pressure. The green solid was collect-
ed by filtration and washed with cold distilled water (2”10 mL) and di-
ethyl ether (2”10 mL). The copper complex was added to a saturated
EDTA disodium salt solution (5 g in 50 mL of distilled water) and the re-
sulting suspension was vigorously stirred at room temperature overnight.
The initial green color disappeared gradually and a yellow solid was sep-
arated. After filtering, washings with cold distilled water (2”10 mL) and
pentane (2”20 mL) and drying under vacuum Ne-Pydec-lysine (20) was
obtained as a yellow solid (0.34 g, 0.95 mmol, 53%). M.p. > 2608C; [a]D20
(t, 3J
A
N
7H), 8.25–8.31 ppm (m, 2H); 13C NMR (75.5 MHz, CDCl3): d=36.7,
66.8, 122.1 (2C), 125.7 (2C), 127.7, 128.4 (2C), 129.5 (2C), 136.8, 145.8,
152.5, 155.7 ppm; IR (KBr): nmax =1075, 1110, 1164, 1218 (broad), 1297,
1347, 1380, 1439, 1477, 1492, 1520, 1594, 1616, 1769, 2859, 2959, 3057,
3081, 3117 cmꢀ1; HPLC (system A): tR =37.7 min, purity 90%; UV/Vis
(recorded during the HPLC analysis): lmax =241, 266 nm; MS (ESI, posi-
tive mode): m/z: calcd for C15H13NO5S2: 351.40; found: 391.50 [M+K]+;
elemental analysis calcd (%) for C15H13NO5S2: C 51.27, H 3.73, N3.99, S
18.25; found: C 51.25, H 3.69, N3.93, S 18.31.
2-(Pyridin-2-yldisulfanyl)ethyl 4-nitrophenyl carbonate (16): 2-(Pyridin-2-
yldisulfanyl)ethanol (12) (0.85 g, 4.5 mmol) was dissolved in dry CH3CN
(13 mL). Triethylamine (0.70 mL, 5.0 mmol) was added and the resulting
mixture was cooled at ꢀ208C. Then, 4-nitrophenylchloroformate (1.05 g,
5.2 mmol) was added and the reaction mixture was stirred at room tem-
perature overnight under N2 atmosphere. The reaction was checked for
completion by TLC (cyclohexane/ethyl acetate 7:3) and the mixture was
evaporated to dryness. The resulting residue was purified by chromatog-
raphy on a silica gel column (50 g) with a step gradient of ethyl acetate
(0!30%) in cyclohexane as the mobile phase, giving activated carbonate
16 as a colorless oil (1.27 g, 3.6 mmol, 78%). Rf =0.43 (cyclohexane/ethyl
=
+12.48 (c=1 in 80% AcOH); 1H NMR (300 MHz, [D6]DMSO): d=
1.38 (m, 4H), 1.60–1.72 (m, 2H), 2.97 (m, 3H), 3.10 (t, 3J
(H,H)=5.9 Hz,
2H), 4.18 (t, 3J
(H,H)=5.9 Hz, 2H), 7.29 (m, 2H), 7.85 (m, 2H),
8.50 ppm (d, 3J(H,H)=4.1 Hz, 1H, NH2); 13C NMR (75.5 MHz,
AHCTREUNG
AHCTREUNG
AHCTREUNG
[D6]DMSO + 5% [D]TFA): d=22.6, 29.8, 30.5, 38.4, 40.5, 52.7, 62.4,
120.6, 122.4, 139.1, 150.4, 156.8, 159.2, 172.0 ppm; IR (KBr): nmax =756,
1144, 1274, 1326, 1419, 1447, 1534, 1576, 1686, 2947, 3342 cmꢀ1; HPLC
(system B): tR =19.2 min, purity 95%; UV/Vis (recorded during the
HPLC analysis): lmax =262 nm; MS (ESI, positive mode): m/z: calcd for
C14H21N3O4S2: 359.47; found: 360.12 [M+H]+, 382.19 [M+Na]+; elemen-
tal analysis calcd (%) for C14H21N3O4S2: C 46.78, H 5.89, N11.69, S
17.84; found: C 46.54, H 5.98, N11.55, S 17.53.
acetate 7:3); 1H NMR (400 MHz, CD2Cl2): d=3.08 (t, 3J
2H), 4.46 (t, 3J
(H,H)=6.3 Hz, 2H), 7.05 (m, 1H), 7.28–7.64 (m, 4H),
8.18 (m, 2H), 8.37 ppm (brd, 3J(H,H)=4.5 Hz, 1H); 13C N MR
A
ACHTREUNG
AHCTREUNG
(100 MHz, CD2Cl2): d=37.2, 67.1, 120.3, 121.5, 122.2 (2C), 125.6 (2C),
137.6, 145.8, 150.1, 152.6, 155.8, 159.6 ppm; IR (neat): nmax =664, 718,
761, 858, 985, 1082, 1112, 1214 (broad), 1346, 1419, 1448, 1522, 1593,
1617, 1764 (broad), 2958, 3081, 3116 cmꢀ1
; HPLC (system A): tR =
Na-(9-Fluorenylmethyloxycarbonyl)-Ne-(2-phenyldisulfanyl-ethyloxycar-
bonyl)-l-lysine (21): Ne-Phdec-lysine 19 (0.37 g, 1.0 mmol) was dissolved
in 4% sodium carbonate solution (6 mL) and cooled to 48C. A solution
of Fmoc-OSu (0.28 g, 0.83 mmol) in 1,4-dioxane (6 mL) was added in one
portion to the stirred solution and the mixture was left at room tempera-
ture overnight. The reaction was checked for completion by TLC
(CH2Cl2/MeOH/AcOH 95:4:1) and the mixture was acidified to pH ~7
with glacial acetic acid. After partial removal of 1,4-dioxane under re-
duced pressure, the reaction mixture was lyophilized. The resulting resi-
due was purified by chromatography on a silica gel column (30 g) with a
step gradient of MeOH (0!5%) in dichloromethane as the mobile
phase. After drying under vacuum, Na-Fmoc-Ne-Phdec-lysine (21) was
obtained as a pale yellow foam (0.30 g, 0.52 mmol, 64%). Rf =0.60
31.6 min, purity 91%; UV/Vis (recorded during the HPLC analysis):
lmax =243, 272 nm; MS (ESI, positive mode): m/z: calcd for
C14H12N2O5S2: 352.39; found: 353.63 [M+H]+; elemental analysis calcd
(%) for C14H12N2O5S2 (contaminated with 6% of 4NP): C 47.96, H 3.44,
N8.07, S 17.11; found: C 47.83, H 3.49, N8.06, S 17.02.
Ne-(2-Phenyldisulfanylethyloxycarbonyl)-l-lysine (19): l-Lysine monohy-
drochloride (0.37 g, 2.0 mmol) was dissolved in distilled water (3 mL) and
basic CuCO3 (i.e., CuCO3·Cu(OH)2·H2O, 0.31 g, 1.3 mmol) was added.
The resulting mixture was refluxed for 2 h and then the hot suspension
was filtered over Celite 545 and washed with distilled water (~20 mL).
After cooling to 48C, the reaction mixture was basified to pH 9–10 with
Na2CO3 (1.08 g, 10.2 mmol) and activated carbonate 15 (0.60 g,
1.7 mmol) dissolved in CH3CN(11 mL) was added. After stirring at
room temperature overnight, a bulky blue precipitate was formed. The
reaction mixture was acidified to pH ~7 with glacial acetic acid and
CH3CNwas removed under reduced pressure. The blue solid was collect-
ed by filtration and washed with cold distilled water (2”10 mL) and di-
ethyl ether (2”10 mL). The copper complex was added to a saturated
EDTA disodium salt solution (5 g in 50 mL of distilled water) and the re-
sulting suspension was vigorously stirred at room temperature overnight.
The initial blue color disappeared gradually and a white solid was sepa-
rated. After filtering and washings with cold distilled water (2”10 mL)
and pentane (2”20 mL), residual water was removed by lyophilization to
give Ne-Phdec-lysine 19 as a white solid (0.45 g, 1.25 mmol, 74%). M.p.
(CH2Cl2/MeOH/AcOH 95:4:1); [a]D20
=
+4.98 (c=1 in CH2Cl2);
1H NMR (300 MHz, CDCl3): d=1.25–1.85 (m, 6H), 2.86–3.13 (m, 4H),
4.19–4.80 (m, 6H), 5.67–5.74 (m, NH), 6.32 (brs, NH), 7.18–7.75 ppm (m,
13H); 13C NMR (75.5 MHz, CDCl3): d=29.6, 31.9, 37.9, 40.7, 47.5, 53.8
(2C), 62.9, 67.4, 120.3 (2C), 125.4, 127.4 (2C), 128.1 (6C), 129.4 (2C),
137.3, 141.6 (4C), 144.0, 144.2, 156.7 ppm; IR (KBr): nmax =739, 1066,
1250, 1449, 1526, 1706 (broad), 2939, 3324 cmꢀ1; HPLC (system C): tR =
30.2 min, purity 92%; UV/Vis (recorded during the HPLC analysis):
lmax =262 nm; MS (ESI, positive mode): m/z: calcd for C30H32N2O6S2:
580.73; found: 581.11 [M+H]+, 1161.04 (dimer) [2M+H]+; elemental
analysis calcd (%) for C30H32N2O6S2·CH4O (MeOH): C 60.76, H 5.92, N
4.57, S 10.47; found: C 60.98, H 5.47, N4.78, S 10.24.
> 2608C; [a]2D0
=
+10.28 (c=1 in AcOH 80%); 1H NMR (300 MHz,
Chem. Eur. J. 2006, 12, 3655 – 3671
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3667

A. Romieu et al.
Na-(9-Fluorenylmethyloxycarbonyl)-Ne-(2-(pyridin-2-yldisulfanyl)ethyl-
oxycarbonyl)-l-lysine (22): Ne-Pydec-lysine 20 (0.31 g, 0.90 mmol) was
dissolved in 4% sodium carbonate solution (5 mL) and cooled to 48C. A
solution of Fmoc-OSu (0.25 g, 0.73 mmol) in a mixture of CH3CN/1,4-di-
oxane 5:1 (6 mL) was added in one portion to the stirred solution and
the mixture was left at room temperature overnight. The reaction was
checked for completion by TLC (CH2Cl2/MeOH/AcOH 90:8:2) and the
mixture was acidified to pH ~7 with glacial acetic acid. After removal of
CH3CNunder reduced pressure, the reaction mixture was lyophilized.
The resulting residue was purified by chromatography on a silica gel
column (40 g, dry loading) with a step gradient of MeOH (0!10%) in
dichloromethane as the mobile phase. After drying under vacuum, Na-
HPLC (system C): tR =33.3 min, purity 93%; MS (ESI, positive mode):
m/z: calcd for C42H48N4O7S2: 785.00; found: 785.3 [M+H]+; elemental
analysis calcd (%) for C42H49N4O7S2·CH4O (MeOH): C 63.21, H 6.42, N
6.86, S 7.85; found: C 63.25, H 6.52, N6.83, S 7.77.
General procedure for the removal of Fmoc-protecting group and tert-
butyl ester: Fmoc-Lys(Ardec)-Phe-OtBu 23 or 24 (0.1 g, 0.12 mmol) was
G
dissolved in CH2Cl2 (2 mL) and cooled to 48C. TFA (1.9 mL) and deion-
ized water (0.1 mL) were added and the resulting reaction mixture was
warmed to room temperature and stirred for 2 h. The reaction was
checked for completion by TLC (CH2Cl2/MeOH 95:5) and the mixture
was evaporated to dryness. TFA was removed by azeotropic distillation
with cyclohexane (3”10 mL). Finally, the product was isolated by chro-
matography on a silica gel column (10–15 g) with a step gradient of
MeOH (0!5%) in dichloromethane as the mobile phase, giving the di-
Fmoc-Ne-Pydec-lysine (22) was obtained as
a yellow foam (0.34 g,
0.58 mmol, 80%). Rf =0.54 (CH2Cl2/MeOH/AcOH 90:8:2); [a]2D0 = ꢀ3.38
(c=1 in CH2Cl2); 1H NMR (300 MHz, CDCl3): d=1.22–1.52 (m, 4H),
peptide Fmoc-Lys
A
1.88 (m, 2H), 2.99 (t, 3J(H,H)=5.6 Hz, 2H), 3.19 (m, 2H), 4.22 (t, 3J-
G
Fmoc-Lys(Ardec)-Phe-OH (40 mg, 0.055 mmol) was dissolved in CH2Cl2
AHCTREUNG
ACHTREUNG
(2 mL). Et2NH (0.17 mL, 1.64 mmol) was added and the resulting reac-
tion mixture was stirred at room temperature overnight. The reaction
was checked for completion by TLC (CH2Cl2/MeOH 95:5) and the mix-
ture was evaporated to dryness. The resulting residue was taken up in a
mixture of aq. TFA and CH3CN4:1 (5 mL) and purified by semi-prepa-
rative RP-HPLC (system D, 2 injections). The appropriate fractions were
A
ACHTREUNG
3.8 Hz, 1H); 13C NMR (75.5 MHz, CDCl3): d=22.1, 29.4, 32.1, 38.3, 40.6,
47.5, 53.9, 63.5, 67.3, 120.3 (2C), 120.6, 121.4, 125.5 (2C), 127.4 (2C),
128.0 (2C), 138.1, 141.6 (4C), 144.1 (2C), 149.3, 156.5, 160.2 ppm; IR
(KBr): nmax =741, 760, 1046, 1082, 1119, 1250, 1418, 1449, 1531, 1715
(broad), 2945, 3065, 3324 cmꢀ1; HPLC (system C): tR =24.9 min, purity
94%; UV/Vis (recorded during the HPLC analysis): lmax =262 nm; MS
(MALDI-TOF, positive mode): m/z: calcd for C29H31N3O6S2: 581.71;
582.3 [M+H]+, 604.2 [M+Na]+, 620.2 [M+K]+ (CHCA matrix); elemen-
tal analysis calcd (%) for C29H31N3O6S2: C 59.88, H 5.37, N7.22, S 11.02;
found: C 59.36, H 5.47, N7.15, S 10.64.
lyophilized, giving the TFA salt of dipeptide H-Lys
white amorphous powder.
A
A
(25):
Yield after RP-HPLC purification: 64%; 1H NMR (300 MHz, CD3CN):
d=1.29–1.46 (m, 4H), 1.81 (m, 2H), 2.95–3.23 (m, 6H), 3.92 (m, 1H, a-
CH Lys), 4.20 (t, 3J
(H,H)=6.0 Hz, 2H), 4.63 (m, 1H, a-CH Phe), 5.73
H
(brs, 1H, NH Phdec), 7.24–7.60 ppm (m, 12H); 13C NMR (75.5 MHz,
CD3CN + 10% H2O): d=21.7, 29.5, 31.2, 37.1, 38.1, 40.6, 53.7, 54.8,
62.7, 118.3, 127.4, 127.9, 128.2 (2C), 129.1 (2C), 129.9 (4C), 137.5, 157.4,
169.4, 173.2 ppm (weak signal); IR (KBr): nmax = 722, 740, 800, 839,
1139, 1203, 1524, 1679 (broad), 2951, 3061 cmꢀ1 (broad); HPLC (system
E): tR =25.9 min, purity 94%; UV/Vis (CH3CN): lmax (e)=237 (6635),
260 nm (1467 molꢀ1 dm3 cmꢀ1); MS (MALDI-TOF, positive mode): m/z:
calcd for C24H31N3O5S2: 505.66; found: 506.4 [M+H]+, 528.4 [M+Na]+,
544.4 [M+K]+ (CHCA matrix); too hygroscopic for elemental analysis.
General procedure for the coupling of Fmoc-Lys(Ardec)-OH to l-phe-
U
nylalanine tert-butyl ester: DIEA (90 mL, 0.51 mmol) was added to a sol-
ution of Na-Fmoc-N e-Ardec-lysine 21 or 22 (0.1 g, 0.17 mmol) and l-
phenylalanine tert-butyl ester hydrochloride (37 mg, 0.17 mmol) dissolved
in dry CH2Cl2 (1–2 mL), followed by BOP coupling reagent (75 mg,
0.17 mmol). The resulting reaction mixture was stirred at room tempera-
ture overnight under N2 atmosphere. The reaction was checked for com-
pletion by TLC (CH2Cl2/MeOH 99:1 or 97:3) and the mixture was evapo-
rated to dryness. The resulting residue was purified by chromatography
on a silica gel column (10–15 g) with a step gradient of MeOH (0!3%)
in dichloromethane as the mobile phase. The appropriate fractions were
pooled and then concentrated to dryness, giving the full-protected dipep-
tide 23 or 24 as white solids.
[Ne-(2-(Pyridin-2-yldisulfanyl)ethyloxycarbonyl]-l-lysyl-l-phenylalanine
(26): Yield after RP-HPLC purification: 60%; 1H NMR (300 MHz,
D2O): d=1.07–1.55 (m, 4H), 1.79 (m, 2H), 2.91–3.26 (m, 6H), 3.89 (t,
3J
8.7 Hz, 3J
3J(H,H)=6.8 Hz, 1H), 8.06–8.19 (m, 2H), 8.51 ppm (brd, 3J
C
ACHTREUNG
Na-(9-Fluorenylmethyloxycarbonyl)-Ne-(2-(2-(phenyldisulfanyl)ethyloxy-
carbonyl)-l-lysyl-l-phenylalanine tert-butyl ester (23): Yield: 80%; Rf =
0.42 (CH2Cl2/MeOH 99:1); 1H NMR (300 MHz, CDCl3): d=1.4 (s, 9H),
1.41–1.85 (m, 4H), 2.87–3.13 (m, 4H), 3.43–3.3.49 (m, 1H), 4.08–4.44 (m,
ACHTREUNG
5.7 Hz, 1H); 13C NMR (75.5 MHz, D2O): d=23.2, 30.7, 32.7, 38.5, 39.9,
42.0, 55.1, 56.7, 64.5, 125.9, 127.1, 129.5, 131.0 (2C), 131.4 (2C), 138.7,
145.6, 146.8, 158.7, 160.2, 171.8, 176.6 ppm; IR (KBr): nmax =702, 723,
764, 800, 839, 1138, 1204, 1261, 1420, 1454, 1538, 1679 (broad), 2944,
3065 cmꢀ1 (broad); HPLC (system E): tR =20.8 min, purity 97%; UV/Vis
(water): lmax (e)=282 (3591), 260 nm (2250 molꢀ1 dm3 cmꢀ1); MS
(MALDI-TOF, positive mode): m/z: calcd for C23H30N4O5S2: 506.65;
found: 507.6 [M+H]+, 569.6 [M+K]+ (CHCA matrix); too hygroscopic
for elemental analysis.
6H), 4.68–4.77 (m, 2H, a-CH Lys and Phe), 5.39 (brd, 3J
NH), 6.30 (brd, 3J
(H,H)=6.4 Hz, 1H), 7.11–7.77 (m, 18H), 8.44 ppm (d,
3J(H,H)=4.9 Hz, 1H); 13C NMR (75.5 MHz, CDCl3): d=29.0 (3C), 29.6,
A
ACHTREUNG
ACHTREUNG
30.9, 37.0, 37.7, 40.9, 47.5, 48.0, 53.5, 63.2, 67.5, 71.4, 120.5 (2C), 125.6
(2C), 127.9 (6C), 128.4 (2C), 128.9 (4C), 129.2 (2C), 132.5, 136.5 (2C),
140.2, 141.9 (2C), 145.5 (2C), 172.0, 174.7 ppm; IR (KBr): nmax =739,
1263, 1537, 1693 (broad), 2939, 3062, 3312 cmꢀ1; HPLC (system C): tR =
34.1 min, purity 95%; MS (MALDI-TOF, positive mode): m/z: calcd for
C43H49N3O7S2: 784.01; found: 806.57 [M+Na]+ (CHCA matrix); elemen-
tal analysis calcd (%)for C43H49N3O7S2·CH4O (MeOH): C 64.76, H 6.55,
N5.15, S 7.86; found: C 64.51, H 6.57, N5.16, S 7.85.
l-Lysyl-l-phenylalanine (28): This compound was prepared from Na-
Fmoc-Ne-Boc-lysine and l-phenylalanine tert-butyl ester hydrochloride
using the classical peptide coupling and deprotection protocols described
for dipeptides 25, 26. Yield after RP-HPLC purification: 40%; 1H N MR
(300 MHz, D2O): d=1.28–1.88 (m, 6H), 2.93 (t, 3J
3.00–3.25 (m, 2H), 3.91 (t, 3J
3J(H,H)=8.8 Hz, 3J
(H,H)=6.0 Hz, 1H, a-CH Phe), 7.27–7.38 ppm (m,
ACHTREUNG
Na-(9-Fluorenylmethyloxycarbonyl)-Ne-(2-(2-(pyridin-2-yldisulfanyl)-
ethyloxycarbonyl)-l-lysyl-l-phenylalanine tert-butyl ester (24): Yield:
95%; Rf =0.26 (CH2Cl2/MeOH 97:3); 1H NMR (300 MHz, CDCl3): d=
1.40 (s, 9H), 1.41–1.85 (m, 4H), 2.97–3.22 (m, 4H), 3.66–3.77 (m, 1H),
AHCTREUNG
A
ACHTREUNG
5H); 13C NMR (75.5 MHz, D2O): d=21.2, 26.6, 30.6, 36.6, 39.3, 53.0,
54.9, 127.5, 129.1 (2C), 129.4 (2C), 136.9, 169.7, 175.2 ppm; IR (KBr):
nmax =724, 802, 841, 1140, 1204, 1437, 1457, 1546, 1679 (broad), 3066 cmꢀ1
(broad); HPLC (system E): tR =12.8 min, purity 97%; UV/Vis (water):
lmax (e)=260 nm (210 molꢀ1 dm3 cmꢀ1); MS (MALDI-TOF, positive
mode): m/z: calcd for C15H23N3O3 293.37; found: 294.4 [M+H]+ (CHCA
matrix); too hygroscopic for elemental analysis.
4.11–4.43 (m, 6H), 4.71 (q, 3J
CH Lys), 4.87 (m, 1H, a-CH Phe), 5.52 (brd, J
(brd, 3J(H,H)=7.4 Hz, 1H), 7.05–7.77 (m, 16H), 8.44 ppm (d, 3J
A
ACHTREUNG
3
AHCTREUNG
A
ACHTREUNG
4.9 Hz, 1H); 13C NMR (75.5 MHz, CDCl3): d=22.3, 29.1 (3C), 29.4, 32.1,
37.1, 38.4, 40.8, 47.6, 54.0, 55.5, 63.5, 67.4, 67.5, 118.6, 121.6, 125.7, 126.5
(2C), 127.8 (6C), 128.4 (2C), 128.9 (2C), 136.5 (2C), 136.7, 140.2, 141.9
(2C), 149.8, 157.5 (2C), 171.7, 174.3 ppm; IR (KBr): nmax =739, 760, 845,
Stability studies of the Ardec groups at the dipeptide level: Solutions of
1152, 1253, 1447, 1522, 1654, 1718 (broad), 2935, 2976, 3312, 3411 cmꢀ1
;
dipeptide
(Ardec) 25 or 26 in dry NMP (1 mgmLꢀ1, 1.6 mm for 25, 1.4 mm
A
3668
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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3655 – 3671

Amine Protecting Groups
FULL PAPER
for 26 or 10 mgmLꢀ1, 16 mm for 25 and 14 mm for 26) were prepared.
Aliquots of the solution (20 mL) were incubated with different amounts
of aq. or organic solutions corresponding to the cleavage conditions re-
ported in Table 1 (entries 1–12), for 1 h at room temperature. Thereafter,
aliquots were quenched with a 1m TFA/NMP solution, diluted with aq.
TFA and analyzed by RP-HPLC (system E). For the stability towards
UV light, 45 mL of the solutions (1 mgmLꢀ1) were transferred into a
quartz fluorescence cell. UV irradiation was performed with a fluores-
cence spectrophotometer by continuous excitation at 350 nm for 1 h at
258C. Thereafter, aliquots were diluted with aq. TFA and anlyzed by RP-
HPLC (system E). Quantification of 25 (or 26) and 28 was achieved by
measurement of the corresponding peak areas at tR =25.9 (or 21.0) and
12.8 min and by using the following extinction coefficients at 260 nm: e=
1467 (25), 2250 (26) and 210 molꢀ1 dm3 cmꢀ1 (28).
Ac-Lys-Asp-Glu-Val-Asp-Lys(Phdec)-NH2 (29): Overall yield after RP-
A
HPLC purification: 17%; HPLC (system G): tR =21.5 min, purity 95%;
MS (MALDI-TOF, positive mode): m/z: calcd for C41H63N9O15S2: 985.38;
found: 986.8 [M+H]+, 1010.8 [M+Na]+, 1024.8 [M+K]+ (CHCA
matrix).
Ac-Lys-Asp-Glu-Val-Asp-Lys(Pydec)-NH2 (30): Overall yield after RP-
G
HPLC purification: 15%; HPLC (system G): tR =16.1 min, purity 93%;
MS (MALDI-TOF, positive mode): m/z: calcd for C40H62N10O15S2:
986.38; found: 987.4 [M+H]+, 1009.4 [M+Na]+, 1025.3 [M+K]+ (CHCA
matrix).
Peptide dimer 31: Overall yield after RP-HPLC purification: 4%; HPLC
(system G): tR =15.9 min, purity 95%; MS (MALDI-TOF, positive
mode): m/z: calcd for C70H116N18O30S2: 1752.75, 1753.9 [M+H]+, 1775.8
[M+Na]+, 1791.8 [M+K]+ (CHCA matrix).
Studies on removal of the Ardec groups at the dipeptide level: Deprotec-
Synthesis and characterization of fluorogenic caspase-3 substrate Ac-Lys-
tion in water: Solutions of dipeptide(Ardec) 25 or 26 in deionized water
A
A
U
(1 mgmLꢀ1, 1.6 mm for 25, 1.4 mm for 26) were prepared. Aliquots of the
solution (10 mL) were incubated with different amounts (5, 10, 20, 40 and
80 equiv) of reducing agent (DTT or b-mercaptoethanol) in solution
(25 mm) in Tris·HCl buffer (0.1m) at pH 7.5, 8.0, 8.5 and 9.0, for 30 min.
Thereafter, aliquots were quenched with aq. TFA and analyzed by RP-
HPLC (system E).
Cy3.0 carboxy succinimidyl ester: Cy3.0 carboxylic acid (3 mg,
4.75 mmol) was introduced into a Reacti-Vial (Pierce, No. 13222) and dis-
solved in dry NMP (75 mL). A solution (75 mL) of TSTU reagent in dry
NMP (1.4 mg, 4.75 mmol) and DIEA (1.65 mL, 9.5 mmol) were added and
the resulting reaction mixture was protected from the light and stirred at
room temperature for 1 h.[61]
Deprotection in organic medium: Solutions of dipeptide(Ardec) 25 or 26
A
Ac-Lys
G
G
in dry NMP (1 mgmLꢀ1, 1.6 mm for 25, 1.4 mm for 26) were prepared.
Aliquots of the solution (10 mL) were incubated with different amounts
(10, 20, 40 and 80 equiv) of a (1:1, mol per mol) b-mercaptoethanol/base
(DBU, DIEA, piperidine and TEA) mixture (25 mm) in dry NMP for
15 min. Thereafter, aliquots were quenched with a solution of TFA in dry
NMP (0.25m), diluted with aq. TFA and analyzed by RP-HPLC (system
E). For further deprotection studies with DBU, aliquots of the solution
were incubated with 80 equiv of b-mercaptoethanol and different
amounts of base (160, 240 and 320 equiv) in solution in dry NMP for
15 min.
Asp-Glu-Val-Asp-Lys
AHCTREUNG
a 1.0 mL Eppendorf tube) was dissolved in dry NMP (130 mL) and DIEA
(4.1 mL, 23.7 mmol) was added. After complete solubilization by vortex-
ing, the resulting solution was added to the crude reaction mixture con-
taining the succinimidyl ester of Cy3.0 dye. The reaction mixture was
protected from the light and stirred at room temperature for 1 h. Finally,
the reaction mixture was quenched by dilution with aq. formic acid
(2 mL) and purified by RP-HPLC (system H, 2 injections). The product-
containing fractions were lyophilized to give the peptide-Cy3.0 conjugate
32 as a pink amorphous powder. Quantification was achieved by UV/Vis
measurements at lmax =549 nm of the Cy3.0 dye by using the e=150000
value (yield after RP-HPLC purification: 60%); HPLC (system I): tR =
26.2 min, purity 95%; UV/Vis (recorded during the HPLC analysis):
lmax =549 nm; MS (MALDI-TOF, positive mode): m/z: calcd for
C71H98N12O22S4: 1598.58; found: 1600.8 [M+H]+, 1621.8 [M+Na]+,
1637.7 [M+K]+ (CHCA matrix).
Solid-phase synthesis of hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys-
A
N
0.52 mmolgꢀ1) was treated with a 20% piperidine solution in NMP
(10 mL) for 20 min. Thereafter, the resin was washed with NMP (2”
10 mL) and CH2Cl2 (2”10 mL). The completion of the Fmoc removal
was checked by the Kaiser ninhydrin test (positive after this treat-
ment).[60] Fmoc-Lys
A
Removal of the Pydec group: Peptide Ac-Lys
G
to the resin with TBTU (0.16 g, 0.5 mmol), HOBt (67 mg, 0.5 mmol) and
DIEA (0.26 mL, 1.5 mmol) in NMP (5 mL) for 2 h (Kaiser ninhydrin test
negative after this time). After washings with NMP (2”10 mL) and 2-
propanol (2”10 mL), the resin was swollen in NMP (1 mL) and transfer-
red into a reaction vessel which was placed in the ABI 433 A peptide
synthesizer. Automated synthesis was carried out according to a general
Lys(Pydec)-NH2 (32) (5.5 mg, 3.2 mmol, weighed in a 2.0 mL Eppendorf
ACHTREUNG
tube) was dissolved in 2.5 mL of Tris·HCl buffer (0.1m, pH 9.0) and a sol-
ution of DTT (25 mg in 1 mL, 160 mmol) in Tris·HCl buffer (0.1m,
pH 9.0) was added. The reaction mixture was protected from the light
and stirred at room temperature for 9 h. The deprotection was checked
for completion by RP-HPLC (system I) and the reaction mixture was
quenched by dilution with aq. formic acid (2 mL). Purification was ach-
ieved by RP-HPLC (system H, 2 injections). The product-containing frac-
Fmoc strategy. Fmoc-Asp
(OtBu)-OH (0.44 g, 1.0 mmol), Fmoc-Val-OH (0.34 g, 1.0 mmol) and
Fmoc-Lys(Boc)-OH (0.47 g, 1.0 mmol) were successively coupled with
A
ACHTREUNG
tions were lyophilized to give the peptide Ac-Lys(Cy3.0)-Asp-Glu-Val-
G
ACHTREUNG
Asp-Lys-NH2 (33) as a pink amorphous powder. Quantification was ach-
ieved by UV/Vis measurements at lmax =549 nm of the Cy3.0 dye by
using the e=150000 value (yield after RP-HPLC purification: 95%);
HPLC (system K): tR =27.2 min, purity 98%; UV/Vis (recorded during
the HPLC analysis): lmax =549 nm; MS (MALDI-TOF, positive mode):
m/z: calcd for C63H91N11O20S2: 1385.58; found 1386.4 [M+H]+, 1408.4
[M+Na]+, 1424.4 [M+K]+ (CHCA matrix).
HBTU (1.0 mmol), HOBt (1.0 mmol) and DIEA (3.7 mmol). Deprotec-
tion of Fmoc groups was carried out with a 20% piperidine solution in
NMP (3”1 min). Acetylation of the free amino group from the N termi-
nal side was achieved by treatment with a mixture of Ac2O (0.43m),
HOBt (0.015m) and DIEA (0.13m) in NMP (5 min). After completion of
the synthetic reaction, the peptide resin Ac-Lys
N
N
A
N
ACHTREUNG
Cy5.0 carboxy succinimidyl ester: Cy5.0 carboxylic acid (1 mg, 1.5 mmol)
was converted into the corresponding succinimidyl ester using the proce-
dure previously described for Cy3.0 dye.
NMP and CH2Cl2 and dried under vacuum (0.25 and 0.2 g, of peptidyl
resins R1 and R2 respectively). Two thirds of the resin was treated with
TFA/PhOH/PhOCH3/H2O 85.8:5.6:4.3:4.3 (10 mL) at room temperature
for 2 h. The filtrate from the cleavage reaction was precipitated with cold
TBME (40 mL) and the peptide was collected by centrifugation. The
white pellet was washed with TBME (2”15 mL) and dried under
vacuum (25 and 38 mg of crude peptides 29 and 30; respectively). The
crude peptide was purified by semi-preparative RP-HPLC (system F, 2
injections). The appropriate fractions were lyophilized, giving the TFA
Ac-Lys
N
N
G
1.0 mL Eppendorf tube) was dissolved in sodium bicarbonate buffer
(300 mL, 0.1m, pH 8.5). After complete solubilization by vortexing, the
resulting solution was added to the crude reaction mixture containing the
succinimidyl ester of Cy5.0 dye. The reaction mixture was protected
from the light and stirred at room temperature for 4 h. Finally, the reac-
tion mixture was quenched by dilution with aq. formic acid (2 mL) and
salt of hexapeptide Ac-Lys-Asp-Glu-Val-Asp-Lys
amorphous powder.
A
Chem. Eur. J. 2006, 12, 3655 – 3671
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3669

A. Romieu et al.
purified by RP-HPLC (system J, 2 injections). The product-containing
fractions were lyophilized to give the peptide Ac-Lys(Cy3.0)-Asp-Glu-
Val-Asp-Lys(Cy5.0)-NH2 34 as a blue-purple amorphous powder. Stock
solution of fluorogenic caspase-3 substrate 34 was prepared in HPLC
grade water and UV/Vis quantification was achieved at lmax =643 nm of
the Cy5.0 dye by using the e=250000 value (yield after RP-HPLC purifi-
cation: 30%); HPLC (system K): tR =36.0 min, purity 95%; UV/Vis (re-
corded during the HPLC analysis): lmax =549, 643 nm; MS (MALDI-
TOF, positive mode): m/z: calcd for C96H129N13O27S4: 2023.80; found:
2025.7 [M+H]+, 2063.6 [M+K]+ (CHCA matrix).
[5] P. M. Collins, R. J. Ferrier, Monosaccharides: Their Chemistry and
Their Roles in Natural Products, Wiley, Chichester, 1995.
[6] N. Sewald, H.-D. Jakubke, Peptides: Chemistry and Biology, Wiley,
Weinheim, 2002.
[7] D. Kadereit, H. Waldmann, Chem. Rev. 2001, 101, 3367–3396.
[8] F. GuibØ, Tetrahedron 1998, 54, 2967–3042.
[9] D. R. Crouch, Tetrahedron 2004, 60, 5833–5871.
[10] a) C. G. Bochet, J. Chem. Soc. Perkin Trans. 1 2002, 125–142;
b) C. G. Bochet, Synlett 2004, 2268–2274.
[11] As illustrated by the thiol-labile cysteine protecting groups for pep-
tide synthesis: 3-nitro-2-pyridinesulfenyl (Npys), S-methyloxycarbo-
nylsulfenyl (Scm), S-[(N-methyl-N-phenylcarbamoylsulfenyl] (Snm)
and S-tert-butylthio (StBu); for a review, see: I. Annis, B. Hargittai,
G. Barany, Methods Enzymol. 1997, 289, 198–221.
[12] a) G. Barany, R. B. Merrifield, J. Am. Chem. Soc. 1977, 99, 7363–
7365; b) G. Barany, F. Albericio, J. Am. Chem. Soc. 1985, 107, 4936–
4942.
[13] M. Planas, E. Bardají, K. J. Jensen, G. Barany, J. Org. Chem. 1999,
64, 7281–7289.
[14] M. J. Barany, R. P. Hammer, R. B. Merrifield, G. Barany, J. Am.
Chem. Soc. 2005, 127, 508–509.
[15] M. Kwiatkowski (Quiatech AB), WO0125247, 2001.
[16] For other examples of 3’-O-blocked fluorescent nucleoside triphos-
phates designed for DNA sequencing by synthesis, see: a) H. Rupar-
el, L. Bi, Z. Li, X. Bai, D. H. Kim, N. J. Turro, J. Ju, Proc. Natl.
Acad. Sci. USA 2005, 102, 5932–5937; b) M. B. Welch, C. I. Marti-
nez, A. J. Zhang, S. Jin, R. Gibbs, K. Burgess, Chem. Eur. J. 1999, 5,
951–960.
[17] C. Schultz, Bioorg. Med. Chem. 2003, 11, 885–898.
[18] J. P. Parrish, R. N. Salvatore, K. W. Jung, Tetrahedron 2000, 56,
8207–8237.
[19] For recent publications reporting the use of a 4-nitrophenyl carbo-
nate derivative to introduce a new N-protecting group on amino
acids, see: a) K. Hojo, M. Maeda, K. Kawasaki, Tetrahedron Lett.
2004, 45, 9293–9295; b) K. Hojo, M. Maeda, K. Kawasaki, Tetrahe-
dron 2004, 60, 1875–1886; c) I. Lingard, G. Bhalay, M. Bradley, Syn-
lett 2003, 1791–1792.
[20] a) H. Salo, A. Guzaev, H. Lçnnberg, Bioconjugate Chem. 1998, 9,
365–371; b) N. Murthy, J. Campbell, N. Fausto, A. S. Hoffman, P. S.
Stayton, Bioconjugate Chem. 2003, 14, 412–419.
AHCTREUNG
ACHTREUNG
In vitro peptide cleavage by recombinant human caspase-3: A 0.53mm
solution of peptide 34 was prepared in caspase-3 buffer (500 mL, 100 mm
NaCl, 40 mm HEPES, 10 mm DTT, 1 mm EDTA, 10% (w/v) sucrose and
0.1% (w/v) CHAPS, pH 7.2) and transferred into a quartz fluorescence
cell. Human recombinant caspase-3 (5 mL, 0.008 U) was added and the
resulting mixture was incubated at 258C. After excitation at 540 nm, fluo-
rescence at 565 nm and 667 nm were simultaneously monitored over time
with measurements recorded every 5 s.
Solid-phase deprotection of the Ardec groups
Application to the synthesis of Ac-Lys-Asp-Glu-Val-Asp-Lys(Ahx-
biotin)-NH2 (35): The peptide resin Ac-Lys
A
G
N
Val-Asp(OtBu)-Lys(Phdec)-Rink amide MBHA (R1) (54 mg, 0.02 mmol)
A
ACHTREUNG
was swollen in dry NMP and treated with a freshly prepared solution of
b-mercaptoethanol (0.5m) and DBU (1.0m) in dry NMP (4.25 mL) for
1 h. Thereafter, the resin was washed with NMP (2”5 mL) and CH2Cl2
(2”5 mL). The completion of the Phdec removal was checked by the
Kaiser ninhydrin test (positive after this treatment). EZ-Link NHS-LC-
biotin (55 mg, 0.10 mmol) was reacted to the free amino group of the
peptide resin R3, in the presence of DIEA (41 mL, 0.23 mmol), in dry
NMP (1.8 mL) overnight (Kaiser ninhydrin test negative after this time).
After washings with NMP (2”5 mL) and CH2Cl2 (2”5 mL) drying under
vacuum, the resin was treated with TFA/PhOH/PhOCH3/H2O
85.8:5.6:4.3:4.3 (5 mL) at room temperature for 2 h. The filtrate from the
cleavage reaction was precipitated with cold TBME (15 mL) and the
peptide was collected by centrifugation. The white pellet was washed
with TBME (2”5 mL) and dried under vacuum. The crude peptide was
purified by semi-preparative RP-HPLC (system L, 2 injections). The ap-
propriate fractions were lyophilized, giving the TFA salt of hexapeptide
Ac-Lys-Asp-Glu-Val-Asp-Lys(Ahx-biotin)-NH2 (35) as
a white amor-
phous powder. Yield after RP-HPLC purification: 40%; HPLC (system
[21] L. Field, T. F. Parsons, D. E. Pearson, J. Org. Chem. 1966, 31, 3550–
3555.
B): tR =18.0 min, purity
> 95%; MS (MALDI-TOF, positive mode):
m/z: calcd for C48H80N12O16S; 1112.55; found: 1113.7 [M+H]+, 1135.7
[M+Na]+, 1151.7 [M+K]+ (CHCA matrix).
[22] D. Moran, K. Sukcharoenphon, R. Puchta, H. F. Schaefer, III,
P. v. R. Schleyer, C. D. Hoff, J. Org. Chem. 2002, 67, 9061–9069.
[23] A. Rosowsky, J. E. Wright, J. Org. Chem. 1983, 48, 1539–1541.
[24] L. Lapatsanis, G. Milias, K. Froussios, M. Kolovos, Synthesis 1983,
671–673.
Acknowledgements
[25] For a recent review on peptide coupling reagents, see: S.-Y. Han, Y.-
A. Kim, Tetrahedron 2004, 60, 2447–2467.
[26] Results from these solution assays are also summarized in a table
available in Supporting Information.
The contributions of Laetitia Bailly (IRCOF/LCOFH), Didier Bertin
(Manteia Predictive Medicine SA), David Chatenet (U 413 INSERM/
IFRMP 23), Colette Lebrun (SCIB/LRI/CEA-Grenoble) and HervØ
Poras (Pharmaleads/UniversitØ Paris 5) to mass spectrometry measure-
ments are greatly acknowledged. We thank Annick Leboisselier
(IRCOF) for the determination of elemental analyses and Dr. CØline
PØrollier (University of Geneva) for NMR experiments of compounds
12, 14 and 16. QUIDD is gratefully acknowledged for generous gift of re-
combinant human caspase-3.
[27] Available pKa data in water at 258C (calculated using Advanced
Chemistry Development (ACD/Labs) Software Solaris V4.67) for 2-
mercaptoethyl methyl carbamate: 9.25 ꢁ 0.25.
[28] D. W. Nicholson, A. Ali, N. A. Thornberry, J. P. Vaillancourt, C. K.
Ding, M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, Y. A. Lazeb-
nik, N. A. Munday, S. M. Raju, M. E. Smulson, T.-T. Yamin, V.-L.
Yu, D. K. Miller, Nature 1995, 376, 37–43.
[29] For reviews on caspases, see: a) J.-B. Denault, G. S. Salvesen, Chem.
Rev. 2002, 102, 4489–4499; b) M. Donepudi, M. G. Grütter, Biophys.
Chem. 2002, 101–102, 145–153; c) H. R. Stennicke, G. S. Salvesen,
Cell Death Differ. 1999, 6, 1054–1059.
[1] T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis,
3rd ed., Wiley, New York, 1999.
[30] S. Kothakota, T. Azuma, C. Reinhard, A. Klippel, J. Tang, K. Chu,
T. J. McGarry, M. W. Kirschner, K. Koths, D. J. Kwiatkowski, L. T.
Williams, Science 1997, 278, 294–298.
[31] For reviews on caspase inhibitors, see: a) M. E. Nuttall, D. Lee, B.
McLaughlin, J. A. Erhardt, Drug Discovery Today 2001, 6, 85–91;
b) R. V. Talanian, K. D. Brady, V. L. Cryns, J. Med. Chem. 2000, 43,
3351–3371.
[2] P. Kocienski, Protecting Groups, 3rd ed., Thieme, Stuttgart, 2003.
[3] P. R. Iyer, S. L. Beaucage in Comprehensive Natural Products Chem-
istry, Vol. 7 (Ed.: E. T. Kool), Pergamon Press, Amsterdam, 1999,
pp. 105–152.
[4] Y. Komatsu, E. Ohtsuka in Comprehensive Natural Products Chem-
istry, Vol. 6 (Eds.: D. Sçll, S. Nishimura, P. B. Moore), Pergamon
Press, Oxford, 1999, pp. 81–96.
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Chem. Eur. J. 2006, 12, 3655 – 3671

Amine Protecting Groups
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Received: December 9, 2005
Published online: March 3, 2006
Chem. Eur. J. 2006, 12, 3655 – 3671
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3671