Diels-alder ligation of peptides and proteins

Article abstract of DOI:10.1002/chem.200600148

The development of the Diels-Alder cycloaddition as a new method for the site-specific chemoselective ligation of peptides and proteins under mild conditions is reported. Peptides equipped with a 2,4-hexadienyl ester and an N-terminal maleimide react in aqueous media to give cycloadducts in high yields and depending on the amino acid sequence with high stereoselectivity. Except for the cysteine SH group the transformation is compatible with all amino acid side chain functional groups. For ligation to proteins the hexadienyl group was attached to avidin and streptavidin noncova-lently by means of complex formation with a biotinylated peptide or by covalent attachment of a hexadienyl ester-containing label to lysine side chains incorporated into the proteins. Site-specific attachment of the hexadienyl unit into a Rab protein was achieved by means of expressed protein ligation followed by protection of the generated cysteine SH by means of Ellman's reagent. The protein reacted with different maleimido-modified peptides under mild conditions to give the fully functional cycloadducts in high yield. The results demonstrate that the Diels-Alder ligation offers an advantageous and technically straightforward new opportunity for the site-specific equipment of peptides and proteins with further functional groups and labels. It proceeds under very mild conditions and is compatible with most functional groups found in proteins. Its combination with other ligation methods, in particular expressed protein ligation is feasible.

Full text of DOI:10.1002/chem.200600148

FULL PAPER
DOI: 10.1002/chem.200600148
Diels–Alder Ligation of Peptides and Proteins
Aline Dantas de Araffljo,[a] Jose M. Palomo,[a] Janina Cramer,[b] Oliver Seitz,[c]
Kirill Alexandrov,[b] and Herbert Waldmann*[a]
Abstract: The development of the
Diels–Alder cycloaddition as a new
method for the site-specific chemose-
lective ligation of peptides and proteins
under mild conditions is reported. Pep-
tides equipped with a 2,4-hexadienyl
ester and an N-terminal maleimide
react in aqueous media to give cycload-
ducts in high yields and depending on
the amino acid sequence with high ster-
eoselectivity. Except for the cysteine
SH group the transformation is com-
patible with all amino acid side chain
functional groups. For ligation to pro-
teins the hexadienyl group was attach-
ed to avidin and streptavidin noncova-
lently by means of complex formation
with a biotinylated peptide or by cova-
lent attachment of a hexadienyl ester-
containing label to lysine side chains
incorporated into the proteins. Site-
specific attachment of the hexadienyl
unit into a Rab protein was achieved
by means of expressed protein ligation
followed by protection of the generat-
ed cysteine SH by means of Ellmanꢀs
reagent. The protein reacted with dif-
ferent maleimido-modified peptides
under mild conditions to give the fully
functional cycloadducts in high yield.
The results demonstrate that the Diels–
Alder ligation offers an advantageous
and technically straightforward new
opportunity for the site-specific equip-
ment of peptides and proteins with fur-
ther functional groups and labels. It
proceeds under very mild conditions
and is compatible with most functional
groups found in proteins. Its combina-
tion with other ligation methods, in
particular expressed protein ligation is
feasible.
Keywords: bioorganic chemistry ·
Diels–Alder reaction ·
ligation techniques
protein modifications
· peptides ·
Introduction
tags is among the most frequently employed and important
methods of research in the life sciences.[1] Recently, in par-
ticular, aldehyde assisted ligations,[2] native chemical liga-
tion,[3] expressed protein ligation,[4] expressed enzymatic li-
gation,[5] Staudinger ligation[6] and the application of the
Huisgen azide cycloaddition[7] have been developed as pow-
erful new methods advancing the field significantly. Howev-
er, due to the multifunctionality of biomacromolecules in
general and proteins in particular and the manifold applica-
tions of such techniques there is a major and continuing
demand for the development of new technology providing
alternatives to the methods mentioned above. The chemistry
required must be compatible with the functional groups
found in proteins and proceed chemoselectively under mild
conditions and in aqueous solution, preferably in the ab-
sence of any potentially denaturating cosolvent. The Diels–
Alder reaction is a highly selective transformation and can
proceed in water with a higher velocity and selectivity than
in organic solvents.[8] Its compatibility with biomolecules has
been explored elegantly in the bioconjugation and/or immo-
bilization of oligonucleotides and other biomolecules[9,10] as
well as microarray development.[11,12] Here we report the de-
velopment of the Diels–Alder cycloaddition as a method for
The site-specific equipment of proteins and other biomole-
cules with additional functional groups for subsequent bio-
chemical and biological investigations, for example, fluoro-
phores, spin probes, photoactivatable groups and affinity
[a] Dr. A. D. de Araffljo, Dr. J. M. Palomo, Prof. H. Waldmann
Department of Chemical Biology
Max-Planck-Institut für molekulare Physiologie
Otto-Hahn-Strasse 11, 44227, Dortmund (Germany)
and
Universität Dortmund, Fachbereich 3, Chemische Biologie
Fax : (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
[b] Dr. J. Cramer, Dr. K. Alexandrov
Department of Physical Biochemistry
Max-Planck-Institut für molekulare Physiologie
Otto-Hahn-Strasse 11, 44227, Dortmund (Germany)
[c] Prof. O. Seitz
Fachinstitut für Organische und Bioorganische Chemie
Institut für Chemie der Humboldt-Universität zu Berlin
Brook-Taylor-Strasse 2(R 2 ’104), 12489 Berlin (Germany)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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the chemoselective ligation of
peptides and proteins under
mild conditions.[13]
Results and Discussion
Scheme 1. Schematic representation of the Diels–Alder ligation of peptides and proteins.
The scope of the ligation ap-
proach was initially investigated
using model peptides equipped with a trans,trans-2,4-hexa-
dienyl group at the C-terminus or with a maleimido dieno-
phile at the N-terminus (Scheme 1). The ligation method
was further employed for the covalent modification of pro-
tein dienyl derivatives with maleimido-labeled probes.
Diels–Alder ligation of peptides
Synthesis of peptide dienyl esters: Due to the acid-sensitivi-
ty of the diene, for the synthesis of the peptide hexadienyl
esters in solution or on the solid phase, base- or mildly acid-
labile protecting groups were applied.
Initially a tripeptide hexadienyl ester, H-Val-Ala-Gly-
OHxd 1, was synthesized in solution by preparing a 9-fluore-
nylmethyloxycarbonyl (Fmoc) tripeptide tert-butyl ester,
cleavage of the tert-butyl ester and re-esterification with
trans,trans-2,4-hexadienol (2; see Scheme S1 in the Support-
ing Information). For longer peptides the re-esterification
proceeded only with unsatisfactory yields, and, therefore,
these peptides were synthesized by means of solid-phase
synthesis using the sulfamylbutyryl linker resin developed
by Backes and Ellman[14] and Fmoc, trityl (Trt), 4-methyltri-
tyl (Mtt) or tert-butylthiol (StBu) groups for protecting N-
terminal and reactive side-chain groups of the amino acids
(Scheme 2). Due to the poor nucleophilicity of the sulfona-
mide function, attachment of the first amino acid may result
in low loading and possible racemization.[15] For this reason,
glycine was selected as the C-terminal amino acid for all
diene–peptide sequences. Still quantitative loading of Fmoc-
glycine was only achieved using a large excess of amino acid
and coupling reagents over extended time (Scheme 2).
After peptide chain elongation on resin employing a 2-
Scheme 2. Solid-phase synthesis of peptide hexadienyl esters. a) Fmoc-
Gly-OH (8 equiv), DIC (8 equiv), N-methylimidazole (8 equiv), DCM/
DMF 4:3, 2”18 h, quantitative loading; b) SPPS: i) 20% piperidine in
DMF, 10 min (2”), ii) Fmoc-AA-OH (4 equiv), HBTU (4 equiv), HOBt
(4 equiv), DIPEA (8 equiv), DMF; c) ICH2CN (25 equiv), DIPEA
(10 equiv), NMP, 24 h; d) i) hexadienol 2 (20 equiv), DMAP (0.5 equiv),
THF, 24 h; ii)(only for 3b) 1% TFA/5% TIS in DCM, 2h; e) 20% piper-
idine in DCM or DMF, 40 min; f) DTT, 0.1m NH4CO3/DMF 5:3, 2.5 h.
DCM=dichloromethane, DMF=dimethylformamide, SPPS=solid-phase
peptide synthesis, NMP=N-methylpyrrolidinone, THF=tetrahydrofuran.
Supporting Information). When necessary, the trityl protect-
ing group was removed by treatment of the cleaved peptide
with 1% trifluoroacetic acid (TFA)/5% triisopropylsilane
(TIS) in dichloromethane for 2h at room temperature. Pep-
tide hexadienyl esters 3a–h were isolated in 43–16% overall
yield after HPLC purification (Table 1). The Fmoc protect-
ing group was removed with 20% piperidine in dichlorome-
thane (DCM) or DMF to give peptides 4a–h after HPLC
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexa-
fluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt)/
N,N-diisopropylethylamine
(DIPEA) coupling protocol,
Table 1. Results for the synthesis of peptide hexadienyl esters 3 and 4.
the sulfonamide linker was acti-
vated by alkylation with iodoa-
cetonitrile/DIPEA. Treatment
of the resin with hexadienol 2/
4-(dimethylamino)pyridine
After cleavage from resin
Peptide sequence
After removal of protecting groups
Diene
Yield [%][a]
Diene
Peptide sequence
Yield [%][a]
3a
3b
3c
3d
3e
3 f
3g
3h
Fmoc-KFmocPFLG
43
20
33
4a
4b
4c
4d
4e
4 f
4g
4h
5
KPFLG
72
51
83
73
80
91
92
35
72
Fmoc-PCStBuSMG
PCStBuSMG
KLGFAG
Fmoc-KFmocLGFAG
Fmoc-KFmocLGKMttAG
Fmoc-KFmocCStBuGVFG
Fmoc-KFmocFPIGLFG
Fmoc-KFmocFPIGLGFG
Fmoc-CStBuGPAG
32
KLGKMttAG
KCStBuGVFG
KFPIGLFG
KFPIGLGFG
CStBuGPAG
KCGVFG
(DMAP) resulted in simultane-
ous release and esterification of
22
16[b]
the protected peptides
3
(Scheme 2). The crude products
were purified by reversed-phase
HPLC to remove excess hexa-
37
[a] Yields are reported after HPLC purification. [b] During assembly of 3g one glycine residue coupling was
dienol and DMAP (Figure S1, not complete resulting in the formation of two products 3 f and 3g in 12and 4% yield, respectively.
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Diels–Alder Ligation
FULL PAPER
purification and lyophilization. Deprotection of the StBu
group was accomplished by reduction of the disulfide bond
of peptide 4e with dithiothreitol (DTT) in ammonium bicar-
bonate medium for 2h, affording deprotected diene 5. Sur-
prisingly, in the case of compound 4d, the Mtt group, which
is known to be a very acid sensitive protecting group for
amines,[16] could not be removed by treatment with 1%
TFA/5% triethylsilane (or TIS) in DCM. If the concentra-
tion of TFA was increased to 5% partial removal of the Mtt
group was observed, however, also considerable decomposi-
tion of the diene occurred. Therefore peptide 4d was subse-
quently employed in Mtt-protected form.
Synthesis of peptide derived dienophiles: The synthesis of
the N-terminal maleimide functionalized peptides 6 was ach-
ieved by means of the Fmoc/tBu solid-phase strategy on a
Wang resin and the HBTU/HOBt/DIPEA coupling protocol
(Scheme 3). In order to investigate a possible influence of
linker length on the course of the ligation reaction N-malei-
mido-glycine[17] or N-maleimido-b-alanine were utilized for
the incorporation of the maleimide group in the last step of
the peptide synthesis. Since the maleimide moiety is stable
under acidic conditions,[17] side chain deprotection and
cleavage were achieved by treatment of the resin with TFA
and scavengers, affording N-maleimido peptides 6a–e in
Scheme 3. Synthesis of the N-terminal maleimido-peptides 6 and Diels–
Alder ligation of the dienyl and maleoyl peptides. a) TFA/DCM 1:1,
80 min; b) dansyl-Cl, NaHCO3, MeOH/H2O 5:2, overnight, 52%; c)
Wang resin, DIC, DMAP, DMF, overnight, quantitative loading; d)
SPPS: i) 20% piperidine in DMF, 10 min (2”); ii) Fmoc-AA-OH
(4 equiv), HBTU (4 equiv), HOBt (4 equiv), DIPEA (8 equiv), DMF; e)
N-maleoyl-glycine (n=1) or N-maleoyl-b-alanine (n=2), DIC, HOBt,
DCM/DMF 1:1; f) TFA/TIS/H2O 95:2.5:2.5, 2–3 h. g) aqueous media,
room temperature. Dansyl=5-dimethylaminonaphthalene-1-sulfonyl.
high purity after lyophilization (Table 2). Fmoc-Lys(dansyl)-
A
OH (7) was applied as building block for the synthesis of
fluorescently labeled peptide 6e.
Table 2. Results for the synthesis of N-maleimido-peptides 6.
Maleimide
Linker (n)
Peptide sequence
Overall yield [%]
6a
6b
6c
6d
6e
1
1
1
1
YTG
TQFHG
SEWIG
62
60
53
59
57
formed in 70% to quantitative yield as revealed by HPLC
analysis. In some cases, consumption of the starting materi-
als was complete only after longer reaction times (Table 3,
entries 4, 6, 7). The use of DMF as a co-solvent seems to
slow down the rate of cycloaddition (Table 3, entry 3 vs 4).
Application of an excess of dienophile over diene considera-
bly shortened the coupling time and led to complete conver-
sion of the hexadienyl peptide to the new cycloadduct
(Table 3, entries 1 and 5).
AKTSAESYSG
2SKTK
A
Peptide ligation by means of Diels–Alder reaction: The
Diels–Alder ligation of the peptide hexadienyl esters 1 or 4
and maleimides 6 to give cycloadducts 8 (Scheme 3) was
performed in aqueous solution at room temperature
(Table 3). The diene and dieno-
phile were mixed in equal
Table 3. Results for the Diels–Alder ligation of hexadiene- and maleimido-peptides.
Entry Cycloadduct Diene Maleimido Solvent Conversion
[h] [%][a]
amounts in most cases and usu-
ally at 10 mm concentration and
allowed to react for 24 h. If re-
quired, methanol or DMF was
added for peptide solubilization
in the aqueous solution.
A typical time course dia-
gram for the Diels–Alder liga-
tion of diene and dienophile
modified peptides is given in
Figure 1. After overnight reac-
t
Isolated after HPLC
[%]
peptide
peptide
1
8a
1
6a[b]
H2O/MeOH
10:3
20
quant.
87
2
3
8b
8c
4a
4d
6a
6b
H2O/MeOH 4:1 24
93
4 95
60
74
H2O/MeOH
20:1
2
4
5
6
7
8d
8e
8 f
8g
4d
4b
4 f
4g
6c
6a[c]
6d
H2O/DMF 4:1
H2O/MeOH 3:422 quant.
H2O
H2O
47
84
64
32
48
48
93
92
69
6d
67
[a] Based on the consumption of diene–peptide determined by analytical HPLC. [b] Dienophile was added in
tion the ligation products were excess (1.2equiv). [c] Dienophile was added in excess (2.4 equiv).
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H. Waldmann et al.
lectivity was scrutinized em-
ploying model compounds ame-
nable to spectroscopic analysis.
The endo/exo selectivity of the
cycloaddition reaction involving
the peptidyl hexadiene and -
maleimide was first investigated
by using simple achiral cycload-
ducts as model molecules (see
Section S4, Supporting Infor-
mation). NOE investigations re-
vealed that only the endo iso-
Figure 1. Time course for the ligation of peptides 4 f (c) and 6d (a) to give cycloadduct 8 f (b) followed by
HPLC analysis (entry 6, Table 3).
All ligation products were isolated by HPLC purification
and identified by mass spectroscopy and NMR (see Section
S3 in the Supporting Information). These results revealed
that the Diels–Alder ligation is chemoselective and compati-
ble with reactive amino acids
mers were formed (Scheme S2, Supporting Information).
The Diels–Alder ligation process to give peptides 8a and
8b led to the formation of the expected endo products
(Figure 2) as confirmed by comparison of their NMR values
such as Lys, His and Trp. Also,
no nucleophilic addition of the
N-terminal amino group to the
a,b-unsaturated double bond of
the dienophile was observed
under these conditions. Howev-
er, we recognized the potential-
ly troublesome Michael addi-
tion reaction that can take
place between the side chain of
cysteine residues and the malei-
mide function.[1] Indeed, when
hexadiene 5, which possesses a
free cysteine residue, was treat-
ed with maleimide 6b, the for-
mation of
a doubly labeled
product resulting from both nu-
cleophilic addition and cycload-
dition reaction was observed
Figure 2. Structures of cycloadducts 8a and 8b.
(68% conversion, MALDI-
TOF: m/z: calcd for: 2142,
found: 2143). To avoid this side
reaction, protection of the cysteine side chain during Diels–
Alder ligation is required, as illustrated for the reaction of
peptides 4b and 6a (Table 3, entry 5), where the sulfhydryl
moiety is masked as a disulfide by the StBu protecting
group.
In additional experiments, we investigated the Diels–
Alder peptide ligation using different diene and dienophile
building blocks. However, peptides incorporating a cyclo-
pentadiene moiety rapidly dimerized in aqueous media[18]
and peptides having a furan group at the C-terminus or an
acrylamide group at the N-terminus, were not reactive
enough.
with the data found for the model cycloadducts (Table S1,
Supporting Information). Surprisingly, cycloadduct 8a was
obtained as a single endo isomer (95%) whereas the cyclo-
adduct 8b was obtained as a mixture of the two endo iso-
mers (50:50). These results suggest that a favourable hydro-
gen-bond pattern between the two reacting peptide chains
may fix the diene and dienophile groups in a specific posi-
tion that favors a face-selective endo attack. Spectroscopic
and chromatographic analysis of the other ligation products
8c–g did not permit a conclusive determination of the ster-
eoselectivity of the cycloaddition processes.
Diels–Alder ligation of proteins
Stereoselectivity of the Diels–Alder ligation: The Diels–
Alder ligation investigated in this study potentially results in
the formation of four diastereomers. Only if endo/exo- and
face-selectivity are complete will one stereoisomer be
formed. In order to investigate this possibility the stereose-
In order to generate tailor-made proteins we sought to com-
bine the Diels–Alder ligation method with other conjugation
techniques. In such a combined strategy the protein of inter-
est is initially functionalized with a diene unit and then the
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Diels–Alder Ligation
FULL PAPER
resulting protein-derived diene can be further functionalized
by Diels–Alder reactions with different dienophiles under
very mild conditions. Initially the Diels–Alder ligation of
proteins was investigated employing a dienyl peptide equip-
ped with a biotin unit that was employed for non-covalent
attachment to streptavidin and avidin. These experiments
revealed that the cycloaddition proceeded smoothly giving
rise to the desired protein cycloadducts (Section S5, Sup-
porting Information).
Dansyl- (12) and fluorescein-maleimide (13) were synthe-
sized from the precursor 2-maleimido-ethanamine[19] as
shown in Scheme 4. The cycloaddition reaction was carried
out at 100 mm protein concentration using 30-fold excess of
dienophile at 258C for 24 h (Scheme 4). After removal of
unligated dienophile, the formation of the new fluorescent
protein 14 was verified by SDS-PAGE for all three conjuga-
tion reactions (Figure 3a). In addition, the mass of the ex-
pected ligation cycloadduct was confirmed by MALDI-TOF
analysis (Supporting Information Figure 4). Incubation of
unmodified streptavidin and dienophile molecules 6e, 12
and 13 resulted in no detectable product (lanes 3, 5 and 7 in
Figure 3a), thus confirming that the observed conjugation of
maleimide probes proceeded via Diels–Alder ligation.
Next we analyzed the efficiency and selectivity of the cy-
cloaddition at different pH values. We chose not to carry
out the reactions at pH above 7 since under these conditions
a nucleophilic addition of amino groups to the dienophile
double bond may take place.[1] The ligation between diene
conjugate 11 and about 100-fold excess of dansylated malei-
mido-peptide 6e was investigated in sodium phosphate
buffer in the pH range of 5.5 to 7.0 for 24 h at 258C. To
monitor possible side reactions, control reactions were per-
formed in which the streptavidin-conjugate was substituted
by unmodified streptavidin. As shown in Figure 3b the
Diels–Alder cycloaddition is selective at pH 5.5 to 6.5 but
looses its selectivity at higher pH. The appearance of a fluo-
rescent band for the reaction of streptavidin with 6e at pH 7
(lane 9 in Figure 3b) indicated that the maleimido-com-
pound also reacts unspecifically with the protein molecule
under these conditions.
Conjugation of proteins by means of Diels–Alder cycloaddi-
tion: For the development of the Diels–Alder bioconjuga-
tion method, we devised the heterobifunctional cross-linker
9, which embodies a 2,4-hexadiene functionality and the N-
hydroxysuccinimidoyl (NHS) group that can react with
amines (Scheme 4).
Cross-linker 9 was prepared in two steps by mono-esterifi-
cation of 2equiv pimelic acid with 1 equiv hexadienol 2 fol-
lowed by transformation of the diene 10 into the N-hydroxy-
succinimidyl ester 9 (Scheme 4). Linker 9 was first attached
to streptavidin molecules by acylation of lysine residues
through the NHS moiety. The molar ratio between the
cross-linker and streptavidin was kept relatively low (6:1) to
prevent multiple labeling of the streptavidin molecules.
Analysis of the MALDI-TOF spectra of modified streptavi-
din revealed that on average each streptavidin subunit was
equipped with one diene-linker (Figure S4B, Supporting In-
formation).
The diene-modified protein 11 was subjected to Diels–
Alder ligation with three different fluorescent labeled malei-
mide compounds 6e, 12 and 13, respectively in water.
Scheme 4. Diels–Alder conjugation of streptavidin. a) Hexadienol 2, DIC, DMAP, THF, overnight, 43%; b) N-hydroxysuccinimide, DIC, DMAP, THF,
overnight, 82%; c) 6 equiv cross linker 9, H2O, 2h, 25 8C, purified by membrane ultracentrifugation (Microcon, 10 kDa cut-off); d) 30 equiv dienophile,
H2O, 24 h, 25 8C, purified by gel filtration (DyeEx spin columns); e) dansyl chloride, DIPEA, DMF, 1 h, 80%; f) fluorescein succinimidyl ester, DIPEA,
DMF, 2.5 h, 46%.
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H. Waldmann et al.
attempted. The method uses a
genetically engineered intein
and a chitin binding domain
(CBD) as fusion partners to ex-
press and purify the desired
Rab thioester.[21,23] In this study,
we employed a Rab protein
from
Canis
familiaris
(Rab7DC3-MESNA thioester
15, see Scheme 5) truncated by
three amino acids.
For the dienyl linker con-
struction, two different cystein-
yl hexadiene linkers 16 and 4h
were synthesized (Section S8,
Supporting Information).
For the construction of the
C-terminally esterified Rab7
protein, Rab7 thioester 15 was
ligated with peptide hexadienyl
esters 16 or 4h under reducing
Figure 3. a) SDS-PAGE analysis of the streptavidin conjugates 14a, 14b and 14c obtained by Diels–Alder liga-
tion of streptavidin-diene 11 (lane 2) and maleimides 6e (lane 4), 12 (lane 6) and 13 (lane 8), respectively.
Control experiments showed that no binding occurs when wild type streptavidin (lane 1) is combined with
maleimides 6e (lane 3), 12 (lane 5) or 13 (lane 7) in water. b) SDS-PAGE analysis for the Diels–Alder conju-
gation of diene 11 and maleimide 6e at pH 5.5 to 7.0. The observed protein bands represent streptavidin subu-
nits as the tetrameric protein complex was denatured upon heating of the sample at 808C for 3 min with dena-
turating loading buffer prior to gel loading. Strep: streptavidin, M: molecular weight marker.
Site-specific labeling of a Rab protein: The Expressed Pro-
tein Ligation (EPL) method[4] is a suitable tool to equip a
given protein with the diene functionality at a specific posi-
tion. By means of this method, the hexadiene group can be
specifically attached to the C-terminus of the protein by re-
action of a recombinant thioester tagged protein with a pep-
tide or amino acid carrying for example a cysteine at the N-
terminus and a hexadienyl unit at the C-terminus. The EPL
process generates a nucleophilic cysteine residue at the liga-
tion site which should be temporary protected (along with
other accessible cysteine side chains present in the protein)
to avoid undesired modification of the mercapto group in
the subsequent reaction with maleimido probes. After the
Diels–Alder ligation process, the masked cysteines are trans-
formed back into the free thiol form.
This approach was successfully implemented using a Rab
protein. The small Rab GTPases are key regulators of vesic-
ular traffic, which act in vesicle budding, docking and fusion
of intracellular peptides.[20] Many processes in which the
Rab proteins are involved are only partially understood,
making differently and site-specifically labeled Rab proteins
versatile probes to address open questions.[21–23]
As part of this proposed methodology for the labeling of
Rab proteins, Ellmannꢀs reagent, 5,5’-dithiobis(2-nitrobenzo-
ic acid) (DTNB)[24] was employed as an appropriate cysteine
blocking agent that efficiently reacts with the mercapto
groups of the protein to form stable disulfide bonds and ren-
ders them inert toward further reactions with maleimides.
To prove the efficiency of Ellmannꢀs reagent to mask the
cysteine residues of Rab proteins, several control experi-
ments were performed in which wild-type Rab7 was treated
with dansylated maleimide 6e with or without prior DTNB
protection (Section S7, Supporting Information).
conditions overnight at 168C and in the presence of GDP
and MgCl2 for stabilization (Scheme 5).[21] An excess of 20
equivalents of the dienyl compound was employed to ensure
complete conversion. Small amounts of the detergent
CHAPS were also included to increase protein and peptide
solubilization under the ligation conditions. The identity of
the ligated products 17a,b was confirmed by ESI-MS (Fig-
ure S6, Supporting Information).
Without intermediate purification, the ligated hexadienyl
protein 17 was directly submitted to cysteine masking with
excess Ellmannꢀs reagent yielding masked protein hexadien-
yl esters 18a and 18b (Scheme 5). These proteins were then
dialyzed against DA buffer (5 mm sodium phosphate pH 6.0,
2 0 mm NaCl, 0.2m m MgCl2, 2 0mm GDP) to remove all
small molecules (MESNA, Ellmannꢀs reagent, dienyl pep-
tide). As determined by mass spectrometry (Figure S6, Sup-
porting Information), the two accessible cysteine residues of
the truncated Rab7 protein 18 were in fact masked by the
disulfide groups. In the case of hexadiene 18b, the reagent
MESNA itself formed a stable disulfide bond with a cysteine
residue of the protein, acting also as a masking agent.
Next the protected hexadienyl Rab7 proteins 18a,b were
subjected to Diels–Alder reaction with peptide-derived di-
enophile 6e and dansyl derivative 12 (Scheme 5). The cyclo-
addition ligation was carried out in buffer at pH 6.0 and at a
protein concentration of approximately 40 mm for 24 h at
room temperature affording fluorescent labeled Rab7 pro-
teins 19a–d. The ligation efficiency varied between 50 and
90% depending on the excess of dienophile employed
(Table S2, Supporting Information). The coupling reactions
were terminated by addition of excess dithiothreitol which
traps the dienophile and simultaneously converts the disul-
fides into unmasked thiols. Because of the release of the
chromogenic thionitrobenzoic acid (TNB) group into solu-
tion, the ligation solution became yellowish at this point.
With a suitable cysteine masking procedure in hands,
functionalization of Rab7 by means of the expressed protein
ligation method to generate the Rab hexadienyl ester was
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Chem. Eur. J. 2006, 12, 6095 – 6109

Diels–Alder Ligation
FULL PAPER
Scheme 5. Combination of expressed protein ligation and Diels–Alder ligation for site-specific modification of the Rab7 protein. a) 0.25 mm thioester
tagged Rab7DC3 15 in 5 mm sodium phosphate buffer pH 7.5, 20 mm NaCl, 20 mm MESNA, 0.4% CHAPS, 10 mm GDP, 0.2m m MgCl2 and 5 mm peptide
16 or 4h, overnight, 168C; b) DTNB, 4 h, 258C, then dialysis against DA buffer (5 mm sodium phosphate buffer pH 6.0, 20 mm NaCl, 0.2m m MgCl2,
20 mm GDP); c) 1 equiv 40 mm Rab7 18 in DA buffer, 100 equiv maleimide 6e or 12, 2 4 h, 2 85C. d) DTT, 2h, 25 8C. MESNA: sodium 2-mercaptoethane
sulfonate, CHAPS: 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate, GDP: guanosine 5’-diphosphate, DA: Diels–Alder.
The three-step ligation process was followed by SDS-
PAGE (Figure 4) and all intermediates and final products
were identified by ESI-MS mass spectrometry (Figure S6,
Supporting Information). In the presence of 100-fold excess
of dienophile the hexadienyl protein is converted into the
expected fluorescent conjugates with high efficiency (90%).
Undesired multiple labeling of Rab7 was not detected at sig-
nificant levels.
The Diels–Alder conjugation of the masked hexadienyl
Rab protein with the dansylated labeled dienophile ren-
dered this protein insoluble in the reaction solution. The for-
mation of aggregates could not be avoided even when the li-
gation was carried out in the presence of cosolvent (glyc-
erol) or detergents (CHAPS, Triton X-100 or Tween-20), or
at lower protein concentration (10–20 mm). This behavior
was also formerly observed during the semisynthesis of lipi-
dated Rab proteins.[21–22]
The purification of the Rab protein 19a was performed
similarly to the procedure described for the isolation of sem-
isynthetic prenylated Rab proteins.[21a] After incubation of
the Rab hexadienyl ester 18a with maleimide 6e, the pro-
tein precipitate was separated by centrifugation and washed
with methanol in order to remove unligated dienophile. The
resulting pellet was dissolved in denaturation buffer contain-
ing 6m guanidinium chloride and DTT (unmasking of the
cysteine at this point) and then refolded by a 25-fold dilu-
tion with renaturation buffer. Subsequently, the folded pro-
tein was dialyzed and concentrated.
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6101

H. Waldmann et al.
subjected to Diels–Alder liga-
tion with BODIPY-labeled pep-
tides.[13]
Thus, upon treatment of pro-
tein dienyl ester 18a with male-
imide-tagged doubly lipidated
peptide 20 the desired lipid-
modified protein 21 was formed
and isolated (Scheme 6 and
Figure 6). The ligated protein
was identified by ESI-MS (m/z:
calcd for: 25496 Da; found:
25495 Da [M+]). Lipidated
peptide 20 represents a fluores-
cent-labeled analogue of the S-
palmitoylated and S-farnesylat-
ed C-terminus of the human
Ras protein and was synthe-
sized as described previously.[27]
Notably the synthesis of chi-
meric lipoprotein 21 also dem-
Figure 4. SDS-PAGE analysis of the Diels–Alder ligation between masked Rab7 hexadienyl ester 18 and male-
imide probes 6e and 12. Diels–Alder labeled Rab7 19a–d appeared as a unique fluorescent protein band at ca
24 kDa. Ligation conditions: 18a and 100-fold 6e (lane 1 in a), 18a and 100-fold 12 (lane 3 in a), 18b and 50-
fold 6e (lane 3 in b), 18b and 100-fold 12 (lane 4 in b).
In order to establish whether the generated protein was
natively folded and functionally active we analyzed its inter-
action with the Rab Escort Protein 1 (REP-1). REP-1 is an
accessory factor that facilitates prenylation of Rab GTPases
by presenting them to Rab geranylgeranyltransferase and
subsequently by delivering them to the target membranes.
Recognition of Rab proteins by REP-1 occurs via a large
protein/protein interface and requires integrity of the Rab
GTPase ternary structure.[25] To analyze the interaction of
Rab7 with REP-1 we took advantage of the dansyl label at-
tached to the flexible C-terminus of Rab7 that enables the
use of fluorescence spectroscopy to monitor the interaction
between both proteins. When the solution of semisynthetic
Rab7 19a excited at 333 nm was titrated with increasing
concentrations of REP-1 a dose dependent and saturable in-
crease of fluorescence emission at 440 nm was observed
(Figure 5). Titration data could be fitted using a quadratic
equation describing the binding curve and were consistent
with a 1:1 stoichiometry and a Kd value of 2n m. This Kd
value is very close to the one obtained earlier with fluores-
cent GDP-bound Rab7.[26] The obtained data show that the
developed procedure yields fully active functionalized Rab7
protein.
Figure 5. Spectrofluorometric titration of 19a with REP-1. Conditions:
258C, 25 mm HEPES pH 7.2, 40 mm NaCl, 2m m MgCl2, 2 mm DTE and
20 mm GDP. The concentrations of 19a complexes were 50 nm. The exci-
tation/emission wavelengths were fixed at 333/440 nm. Data were fitted
to a quadratic equation as implemented in the program Grafit 5.1 (Eri-
thacus software) leading to Kd value of 1.9 ꢀ 0.5 nm and consistent with
1:1 stoichiometry. DTE: dithioerythritol, HEPES: 2-[4-(2-hydroxyethyl)-
1-piperazinyl]ethanesulfonic acid.
onstrates that the Diels–Alder ligation is applicable to the
synthesis of sensitive protein conjugates such acid- and
base-sensitive lipoproteins.[28]
The use of the two-step ligation strategy delineated above
is particularly indicated if reporter groups or tags need to be
introduced into proteins that are not stable under the condi-
tions of expressed protein ligation. For instance, in the
course of a different research program we observed that the
fluorescence of the BODIPY label, one of the most advan-
tageous and very often used fluorophores, is lost during at-
tempted ligation of BODIPY-functionalized peptides to pro-
teins by means of EPL. However, fluorescent-labeled Rab
proteins are formed if diene-modified Rab protein 18a is
Conclusion
We have shown that the Diels–Alder ligation offers a new
opportunity for the site-selective functionalization of pro-
teins and peptides. It proceeds under very mild conditions
with high selectivity and is compatible with most functional
groups found in proteins. Its combination with other ligation
methods, in particular expressed protein ligation, is feasible
and allows for the rapid equipment of a given protein with
different functional groups in a site-specific manner, that is
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Chem. Eur. J. 2006, 12, 6095 – 6109

Diels–Alder Ligation
FULL PAPER
Scheme 6. Combination of expressed protein ligation and Diels–Alder ligation for the synthesis of a lipidated protein. a) 1 equiv 40 mm Rab7 18a in DA
buffer, 100 equiv maleimide 20 (in MeOH/CH2Cl2 3:1), 24 h, 258C (ligated product 21 precipitates); b) centrifugation and washing of the pellet with
MeOH (3”) and to remove excess peptide; c) the precipitate was dissolved in denaturation buffer (100 mm Tris-HCl, pH 8.0, 6m guanidinium-HCl,
100 mm DTE, 1% CHAPS, 1 mm EDTA) over night at 48C, d) protein renaturated by diluting 25-fold dropwise into refolding buffer (50 mm HEPES,
pH 7.5, 2.5 mm DTE, 2m m MgCl2, 10 mm GDP, 1% CHAPS, 400 mm arginine-HCl, 400 mm trehalose, 0.5 mm PMSF, 1 mm EDTA) 20-fold stirring at
258C 3 h, e) protein was concentrated up to ca. 40 mm by ultracentrifugation (Amicon 10 kDa cut-off). Tris: tris(hydroxymethyl)aminomethane, EDTA:
ethylenediaminetetraacetate, PMSF: phenylmethylsulfonyl fluoride.
hexadienol. Maleimide derivatives and probes are available
from commercial suppliers.
Taken together the results described herein and the previ-
ous results for the bioconjugation of oligonucleotides and
saccharides demonstrates that the Diels–Alder [4+2] cyclo-
addition is an advantageous method for covalent biomole-
cule modification and a real alternative for the in vitro as-
sembly of semisynthetic proteins and biopolymers.
Experimental Section
Figure 6. SDS-PAGE analysis of the Diels–Alder ligation product 21
(lane 3) obtained from masked Rab7 hexadienyl ester 18a (lane 1) and
maleimide BODIPY-peptide 20 (lane 2).
General methods: Peptide synthesis grade solvents and deionized water
(Millipore Q-Plus System) were used for all experiments. Peptides and
reagents, unless otherwise noted, were purchased from commercial sup-
pliers. Purifications were performed by reversed-phase HPLC on Agilent
preparative HPLC 1100 Series system using a Nucleodur C18 Gravity
column (Macherey–Nagel) and detection at 215 nm. Linear gradients of
solvent B (0.1% TFA in acetonitrile) in solvent A (0.1% TFA in water)
were applied at 25 mLminꢁ1 flow rate. Optical rotations were measured
in a Schmidt and Haensch Polartronic HH8 polarimeter at 589 nm and
concentrations are given in g per 100 mL solvent. NMR spectra were re-
corded using a Varian Mercury 400 MHz spectrometer and calibrated ac-
cording with solvent standard peaks. Analytical reversed-phase HPLC
was carried out in a Hewlett Packard HPLC 1100 system using a Nucleo-
syl 100-5 C18 Nautilus column, detection at 215 and 254 nm and linear
gradients of solvent B in solvent A at 1 mLminꢁ1 flow rate. Electrospray
mass spectrometric analyses (ESI-MS) were performed on a Finnigan
LCQ spectrometer. Mass spectra from proteins were deconvoluted using
the Biomass program included in the Xcalibur software (ThermoFinni-
gan) Fast atom bombardment (FAB) mass spectra were recorded on a
at the C-terminus. The Diels–Alder coupling involving the
maleimide segment should be carried out at slightly acidic
conditions and in the absence of reactive thiol groups or
other groups of similar nucleophilicity. If the protein pos-
sesses reactive cysteine residues in its structure, the ligation
conditions must be adjusted to prevent unspecific reactions
by temporary blocking of the sulfhydryl groups. The Diels–
Alder reaction leads to a non-traceless ligation site, but the
final cycloadduct skeleton is relatively small and should not
significantly influence the protein structure. The 2,4-hexa-
diene moiety is stable under physiological conditions and
can be easily incorporated chemically into biomolecules
from the commercially available precursor trans,trans-2,4-
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6103

H. Waldmann et al.
Finnigan MAT MS 70 spectrometer, using m-nitrobenzylalcohol as
matrix. MALDI-TOF measurements were carried out with Voyager-DE
Pro Biospectrometry Workstation from PerSeptive Biosystems utilizing
a-cyano-hydroxycinnammic acid as matrix. Calculated masses were ob-
tained using the program ChemDraw Ultra (CambridgeSoft Corpora-
tion). Streptavidin and avidin proteins were obtained from Sigma. Tris/
glycine 15% gels were applied for denaturating SDS-PAGE, with fluores-
cence detection at 302nm (Reprostar II UV illuminator, Camag) and
subsequent staining with Coomassie Blue. Rab7 thioester,[21] and REP-
1[25] were expressed and purified as described in the respective referen-
ces.
(s, 3H, CH3 Met), 2.10–2.38 (m, 2H, b-CH2 Met), 2.44–2.65 (m, 2H, g-
CH2 Met), 2.92–3.20 (m, 2H, b-CH2 Cys), 3.41–3.70 (m, 2H, e-CH2 Pro),
3.78–3.88 (m, 2H, b-CH2 Ser), 3.92(m, 2H, a-CH2 Gly), 4.18–4.56 (m,
6H, CH Fmoc, CH2 Fmoc, 3”a-CH), 4.59 (d, 2H, J=6.5 Hz, CH2 Hxd),
4.62–4.66 (m, 1H, a-CH), 5.57–5.63 (m, 1H, CH=CHCH3), 5.70–5.79 (m,
1H, CH2CH=CH), 6.01–6.08 (dd, 1H, J=15.0, 10.4 Hz, CH=CHCH3),
6.21–6.28 (dd, 1H, J=15.0, 10.3 Hz, CH2CH=CH), 7.32(t, H2 ,
J=
7.4 Hz, Ar Fmoc), 7.40 (t, 2H, J=7.4 Hz, Ar Fmoc), 7.65 (t, 2H, J=
7.4 Hz, Ar Fmoc), 7.80 ppm (d, 2H, J=7.4 Hz, Ar Fmoc) and weak
amide signals; ESI-MS: m/z for C43H57N5O9S3: 884.3; found: 884.4
[M+H]+; MALDI-TOF: m/z: 906.8 [M+Na]+, 922.8 [M+K]+.
General procedure for solid-phase peptide synthesis (SPPS)
Peptides were assembled by manual solid-phase synthesis using the
HBTU/HOBt/DIPEA activation method. Typically couplings were car-
ried out with 4 equiv Fmoc-amino acid, 4 equiv HBTU, 4 equiv HOBt
and 8 equiv DIPEA in DMF, for a minimum of 1 h and monitored by the
Kaiser test. Fmoc-deprotection was accomplished by treating the resin
with 20% piperidine in DMF for 10 minutes twice. N-Maleimidoyl gly-
cine[17] or N-maleimido-b-alanine were coupled using 4 equiv of the re-
spective maleimide, 4 equiv DIC and 4 equiv HOBt in DCM/DMF 1:1
for 2h or allowed to react overnight.
Fmoc-Lys(Fmoc)-Leu-Gly-Phe-Ala-Gly-OHxd (3c): Starting from sulfo-
A
namide resin (51 mg) loaded with Fmoc-Gly (0.043 mmol) a colorless
solid (16 mg, 0.014 mmol, 33%) was obtained. 1H NMR ([D6]DMSO,
400 MHz): d=0.81 (d, 3H, J=6.5 Hz, CH3 Leu), 0.82(d, 3H, J=6.5 Hz,
CH3 Leu), 1.23 (d, 3H, J=7.1 Hz, CH3 Ala), 1.22–1.65 (m, 9H, 3”CH2
Lys, b-CH2 Leu, g-CH Leu), 1.70 (d, 3H, J=6.6 Hz, CH3 Hxd), 2.71–2.77
(m, 1H, b-CH2 Phe), 2.92–2.99 (m, 2H, CH2 Lys), 2.99–3.05 (m, 2H, b-
CH2 Phe), 3.55–3.71 (m, 2H, a-CH2 Gly), 3.77–3.91 (m, 2H, a-CH2 Gly),
3.94–4.00 (m, 1H, a-CH), 4.14–4.35 (m, 8H, 2”CH Fmoc, 2” a-CH, 2”
CH2 Fmoc), 4.51–4.55 (m, 1H, a-CH), 4.56 (d, 2H, J=6.2Hz, CH 2 Hxd),
5.60 (ddd, 1H, J=12.8, 6.4, 6.4 Hz, CH=CHCH3), 5.69–5.78 (m, 1H,
CH2CH=CH), 6.04 (dd, 1H, J=14.9, 10.2Hz, C H=CHCH3), 6.27 (dd,
1H, J=15.0, 10.3 Hz, CH2CH=CH), 7.13–7.26 (m, 5H, Ar Phe), 7.31 (t,
4H, J=7.4 Hz, Ar Fmoc), 7.40 (t, 4H, J=7.4 Hz, Ar Fmoc), 7.66–7.71
(m, 4H, Ar Fmoc), 7.84–7.86 (m, 1H, NH), 7.87 (d, 4H, J=7.4 Hz, Ar
Fmoc), 7.98 (d, 1H, J=8.2Hz, NH), 8.05 (t, 1H, J=5.3 Hz, NH), 8.13 (t,
1H, J=5.7 Hz, NH), 8.19 ppm (d, 1H, J=7.6 Hz, NH); ESI-MS: m/z:
calcd for C64H73N7O11: 1116.5; found: 1116.2[ M+H]+; MALDI-TOF:
m/z: 1155.1 [M+K]+.
General procedure for the solid-phase synthesis of the fully protected
hexadienyl peptide esters 3 using the sulfonamide linker resin
First amino acid loading and peptide assembly: The resin loaded with
Fmoc-glycine was obtained by treatment of 4-sulfamylbutyryl AM resin
(Novabiochem) with a mixture of Fmoc-Gly-OH (7 equiv), N-methylimi-
dazole (7 equiv) and DIC (7 equiv) in DCM/DMF 4:3 overnight (quanti-
tative loading as determined by the UV-Fmoc method). The peptide
chain was assembled as indicated in the general procedure above.
Activation/cleavage from resin: Iodoacetonitrile (25 equiv) and DIPEA
(10 equiv) were dissolved in NMP (4 mLmmolꢁ1 ICH2CN), filtered
through basic alumina, and added to the fully protected peptidyl resin
(pre-swollen in DCM and NMP). The resulting mixture was shielded
from light and shaken at room temperature for 18–24 h. The resin was
washed with NMP (5”) and THF (3”). The activated resin was directly
transferred to a round bottom flask and treated with a solution of trans-
trans-2,4-hexadien-1-ol (2; 20 equiv) and DMAP (0.5 equiv) in dry THF
(10 mLgꢁ1 resin) for 24 h. The resin was filtered and washed several
times with THF. The filtrates were combined and THF was removed
under reduced pressure. The crude product was purified by reversed-
phase HPLC. Fractions containing the product (analyzed by MALDI-
TOF) were combined and dried by lyophilization.
Fmoc-Lys
C
G
sulfonamide resin (59 mg) loaded with Fmoc-Gly (0.05 mmol) a colorless
solid (21 mg, 0.016 mmol, 32%) was obtained. ESI-MS: m/z: calcd for
C81H92N8O11: 1353.7; found: 1353.4 [M+H]+; FAB-LRMS: m/z: 1352.79
[M]+. Compound 3d was not further characterized and directly convert-
ed into compound 4d.
Fmoc-Lys
U
Fmoc-Gly sulfonamide resin (164 mg) loaded with Fmoc-Gly
(0.056 mmol), a colorless solid (14 mg, 0.012mmol, 22%) was obtained.
[a]2D0 =ꢁ13.5 (c=0.2, MeOH); 1H NMR ([D7]DMF, 400 MHz): d=0.92
(d, 3H, J=6.8 Hz, CH3 Val), 0.94 (d, 3H, J=6.8 Hz, CH3 Val), 1.46 (s,
9H, tBu), 1.63–1.72(m, 4H, g-CH2 Lys, d-CH2 Lys), 1.88 (d, 3H, J=
6.6 Hz, CH3 Hxd), 1.90–2.10 (m, 2H, b-CH2 Lys), 2.16–2.25 (m, 1H, b-
CH Val), 3.28–3.50 (m, 6H, e-CH2 Lys, b-CH2 Phe, b-CH2 Cys), 4.00–4.21
(m, 4H, 2” a-CH2 Gly), 4.34–4.48 (m, 8H, 2”CH Fmoc, 2” a-CH, 2”
CH2 Fmoc), 4.78 (d, 2H, J=6.4 Hz, CH2 Hxd), 4.85–4.90 (m, 2H, 2” a-
CH), 5.78–5.85 (m, 1H, CH=CHCH3), 5.90–5.98 (m, 1H, CH2CH=CH),
6.22–6.28 (dd, 1H, J=15.0, 11.0 Hz, CH=CHCH3), 6.45–6.52(dd, 1H, J=
15.0, 10.4 Hz, CH2CH=CH), 7.34 (t, 1H, J=7.2Hz, Ar Phe), 7.41 (t, 2H,
J=7.2Hz, Ar Phe), 7.47 (d, 2H, J=7.0 Hz, Ar Phe), 7.50 (t, 4H, J=
7.4 Hz, Ar Fmoc), 7.60 (t, 4H, J=7.4 Hz, Ar Fmoc), 7.84–7.97 (m, 8H,
Ar Fmoc, 4”CONH), 8.05 (d, 4H, J=7.4 Hz, Ar Fmoc), 8.35 (t, 1H, J=
6.0 Hz, CONH), 8.49 (t, 1H, J=5.8 Hz, CONH), 8.66 ppm (d, 1H, J=
7.5 Hz, CONH); ESI-MS: m/z: calcd for C67H79N7O11S2: 1222.5; found:
1222.2 [M+H]+; FAB-LRMS: m/z: 1221.7 [M]+.
Fmoc-Lys(Fmoc)-Pro-Phe-Leu-Gly-OHxd (3a): Starting from sulfona-
A
mide resin (122 mg) loaded with Fmoc-Gly (0.075 mmol), a colorless
solid (35 mg, 0.032mmol, 43%) was obtained. 1H NMR (CDCl3,
400 MHz): d=0.62–0.80 (m, 6H, 2”CH3 Leu), 1.10–2.10 (m, 16H, d-CH2
Lys, g-CH2 Lys, b-CH2 Leu, g-CH Leu, CH3 Hxd, b-CH2 Lys, b-CH2 Pro,
g-CH2 Pro), 2.9–3.2 (m, 4H,b-CH2 Phe, e-CH2 Lys), 3.45–3.70 (m, 2H, e-
CH2 Pro), 3.75–3.90 (m, 2H, a-CH2 Gly), 4.00–4.55 (m, 12H, a-CH Lys,
a-CH Pro, a-CH Leu, a-CH Phe, CH2 Hxd, 2”CH2 Fmoc, 2”CH
Fmoc), 5.36–5.49 (m, 1H, CH=CHCH3), 5.60–5.68 (m, 1H, CH2CH=
CH), 5.82–5.94 (m, 1H, CH=CHCH3), 6.06–6.13 (m, 1H, CH2CH=CH),
7.00–7.10 (m, 5H, Ar Phe), 7.13–7.23 (m, 4H, 2”Ar Fmoc), 7.25–7.35
(m, 4H, 2”Ar Fmoc), 7.40–7.55 (m, 4H, 2”Ar Fmoc), 7.60–7.70 ppm
(m, 4H, 2”Ar Fmoc); ESI-MS: m/z: calcd for C64H72N7O10: 1085.5;
found: 1085.3 [M+H]+.
Fmoc-Lys
Lys(Fmoc)-Phe-Pro-Ile-Gly-Leu-Gly-Phe-Gly-OHxd (3g): Sulfonamide
AHCTREUNG
Fmoc-Pro-Cys
mide resin (151 mg) loaded with Fmoc-Gly (0.10 mmol) the product was
cleaved from the resin as Fmoc-Pro-Cys(StBu)-Ser(Trt)-Met-Gly-OHxd
G
resin (106 mg) loaded with Fmoc-Gly (0.09 mmol) was used. The cou-
pling of the second glycine residue was incomplete, yielding a mixture of
two peptides 3 f and 3g which were separated by means of HPLC: 16%
overall yield: 3 f (14.5 mg, 0.010 mmol, 12%) and 3g (5.4 mg,
and purified by RP-HPLC. The resulting fractions containing product
were combined and concentrated in vacuo. After evaporation of the sol-
vent, partial Trt deprotection was detected (MALDI and LC-MS). In
order to achieve complete removal of the Trt group, the crude product
(58 mg) was further treated with DCM/TFA/TIS 100:1:5 (1 mL) for 2h
at room temperature. Solvents were removed in vacuo and the product
was purified by RP-HPLC, affording colorless solid (18 mg, 0.02mmol,
20%); 1H NMR (CD3OD, 400 MHz): d = 1.20/1.32 (s, 9H, tBu), 1.73 (d,
3H, J=6.6 Hz, CH3 Hxd), 1.86–2.10 (m, 4H, g-CH2 Pro, b-CH2 Pro), 2.06
0.0037 mmol, 4%). 3 f: MALDI-TOF: m/z: calcd for C81H95NaN9O13
:
1424.7; found: 1424.7 [M+Na]+, 1440.7 [M+K]+; 3g: MALDI-TOF:
m/z: calcd for C83H98N10O14: 1481.7; found: 1481.7 [M+Na]+, 1497.7
[M+K]+. Compounds 3 f and 3g were not further characterized and im-
mediately converted to compounds 4 f and 4g.
Fmoc-Cys
A
resin (291 mg) loaded with Fmoc-Gly (0.24 mmol) was used. The product
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Chem. Eur. J. 2006, 12, 6095 – 6109

Diels–Alder Ligation
FULL PAPER
was purified by flash chromatography, eluting with DCM and DCM/
15.0, 10.4 Hz, CH=CHCH3), 6.28 (dd, 1H, J=15.0, 10.6 Hz, CH2CH=
CH), 7.18 (d, 2H, J=8.4 Hz, Ar Mtt), 7.27–7.33 (m, 6H, Ar Mtt), 7.43–
7.49 ppm (m, 6H, Ar Mtt); FAB-HRMS: m/z: calcd for C34H52N6O6:
909.5602; found 909.5684 [M+H]+.
MeOH 100:1!20:1 to give a colorless solid (70 mg, 0.09 mmol, 37%). Rf
=
0.31 (DCM/MeOH 10:1); MALDI-TOF: m/z: calcd for
C40H51NaN5O8S2: 816.3; found: 816.6 [M+Na]+, 833.5 [M+K]+. Com-
pound 3h was not further characterized and immediately converted to
compound 4h.
H-Lys-Cys
C
=
General procedure for Fmoc-protecting group removal to give hexadien-
yl esters 4: A solution of 5–30 mg Fmoc-protected peptide 3 was treated
with 0.5–2.0 mL 20% piperidine in DMF or DCM for 40 minutes at
room temperature. The reaction mixture was coevaporated with metha-
nol to remove excess of piperidine. The product was purified by re-
versed-phase-HPLC, fractions containing the product (evaluated by
MALDI-TOF) were combined and dried by lyophilization.
ꢁ9.08(c=0.1, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.73 (d, 3H, J=
6.8 Hz, CH3 Val), 0.82(d, 3H, J=6.8 Hz, CH3 Val), 1.34 (s, 9H, tBu),
1.45–1.55 (m, 2H, g-CH2 Lys), 1.63–1.70 (m, 2H, d-CH2 Lys), 1.74 (d,
3H, J=6.6 Hz, CH3 Hxd), 1.84–1.95 (m, 2H, b-CH2 Lys), 2.01–2.10 (m,
1H, b-CH Val), 2.94 (t, 2H, J=7.6 Hz, e-CH2 Lys), 2.98–3.07 (m, 2H, b-
CH2 Phe), 3.20 (dt, 2H, J=13.9, 6.4 Hz, b-CH2 Cys), 3.68 (d, 1H, J=
16.1 Hz, a-CH Val), 3.94 (s, 2H, a-CH2 Gly), 3.98 (t, 1H, J=6.4 Hz, a-
CH Lys), 4.08 (t, 2H, J=8.2Hz, a-CH2 Gly), 4.63 (d, 2H, J=6.6 Hz,
CH2 Hxd), 4.63–4.66 (m, 2H, a-CH Cys, a-CH Phe), 5.58–5.65 (m, 1H,
CH=CHCH3), 5.72–5.82 (m, 1H, CH2CH=CH), 6.03–6.10 (dd, 1H, J=
15.0, 10.4 Hz, CH=CHCH3), 6.25–6.32 (dd, 1H, J=15.1, 10.5 Hz,
CH2CH=CH), 7.18–7.30 ppm (m, 5H, Ar Phe); MALDI-TOF: m/z: calcd
for C37H59N7O7S2: 778.4; found 778.8 [M+H]+, 800.8 [M+Na]+, 816.7
[M+K]+.
H-Lys-Pro-Phe-Leu-Gly-OHxd (4a): Starting from 3a (33 mg,
0.032mmol) a colorless solid (15 mg, 0.023 mmol, 72%) was obtained.
[a]2D0 =ꢁ27.98 (c=0.3, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.91
(d, 3H, J=6.1 Hz, CH3 Leu), 0.94 (d, 3H, J=6.2Hz, CH 3 Leu), 1.46–
1.82(m, 7H, d-CH2 Lys, g-CH2 Lys, b-CH2 Leu, g-CH Leu), 1.74 (d, 3H,
J=6.7 Hz, CH3 Hxd), 1.84–2.23 (m, 6H, b-CH2 Pro, g-CH2 Pro, b-CH2
Lys), 2.90–3.00 (m, 2H, e-CH2 Lys), 3.02(dd, 1H, J=13.9, 7.7 Hz, b-CH2
Phe), 3.13 (dd, 1H, J=13.9, 6.5 Hz, b-CH2 Phe), 3.56–3.63 (m, 1H, e-CH2
Pro), 3.67–3.73 (m, 1H, e-CH2 Pro), 3.81–3.96 (m, 2H, a-CH2 Gly), 4.25
(t, 1H, J=6.1 Hz, a-CH), 4.42(dd, 1H, J=9.5, 5.5 Hz, a-CH), 4.50 (t,
1H, J=5.2Hz, a-CH), 4.57 (t, 1H, J=6.6 Hz, a-CH), 4.61 (d, 2H, J=
6.6 Hz, CH2 Hxd), 5.61 (ddd, 1H, J=13.5, 6.5, 6.5 Hz, CH=CHCH3),
5.72–5.81 (m, 1H, CH2CH=CH), 6.06 (dd, 1H, J=15.0, 10.4 Hz, CH=
CHCH3), 6.28 (dd, 1H, J=15.0, 10.4 Hz, CH2CH=CH), 7.17–7.30 ppm
(m, 5H, Ar Phe); ESI-MS: m/z: calcd for C34H52N6O6: 641.4; found:
641.4 [M+H]+; FAB-HRMS: m/z: 640.3945 [M]+.
H-Lys-Phe-Pro-Ile-Gly-Leu-Phe-Gly-OHxd (4 f): Starting from 3 f
(14.5 mg, 10.3 mmol) a colorless solid (9.0 mg, 9.4 mmol, 91%) was ob-
1
tained. [a]2D0 =ꢁ28.68 (c=0.3, MeOH); H NMR (CD3OD, 400 MHz): d=
0.82(dd, 3H, J=6.3, 4.3 Hz, CH3 Ile), 0.87 (d, 3H, J=6.3 Hz, CH3 Ile),
0.91–1.00 (m, 6H, 6”CH3 Leu), 1.21–1.71 (m, 10H, CH2 Ile, d-CH2 Lys,
g-CH2 Lys, b-CH2 Leu, b-CH2 Lys), 1.74 (d, 3H, J=6.6 Hz, CH3 Hxd),
1.83–2.14 (m, 6H, b-CH2 Pro, g-CH2 Pro, g-CH Leu, b-CH Ile), 2.90–3.04
(m, 4H, 2”1Hb-CH2 Phe, e-CH2 Lys), 3.17–3.26 (m, 2H, 2”1H b-CH2
Phe), 3.32–3.52 (m, 1H, e-CH2 Pro), 3.76–3.95 (m, 6H, e-CH2 Pro, 2” a-
CH2 Gly), 4.08 (t, 1H, J=7.0 Hz, a-CH Pro), 4.22–4.28 (m, 2H, 2”a-
CH), 4.53–4.67 (m, 2H, 2” a-CH), 4.62(d, 2H, J=6.5 Hz, CH2 Hxd),
5.61 (ddd, 1H, J=13.6, 6.4, 6.4 Hz, CH=CHCH3), 5.72–5.81 (m, 1H,
CH2CH=CH), 6.06 (dd, 1H, J=15.0, 10.5 Hz, CH=CHCH3), 6.28 (dd,
1H, J=15.2, 10.5 Hz, CH2CH=CH), 7.17–7.34 ppm (m, 10H, Ar Phe);
ESI-MS: m/z: calcd for C51H75N9O9: 958.6; found: 958.7 [M+H]+;
MALDI-TOF: m/z: 958.9 [M+H]+, 980.9 [M+Na]+, 996.8 [M+K]+;
FAB-LRMS: m/z: 958.75 [M+H]+.
H-Pro-Cys
C
ꢁ17.18 (c=0.1, MeOH); 1H NMR (CD3OD, 400 MHz): d=1.35 (s, 9H,
tBu), 1.75 (d, 3H, J=6.6 Hz, CH3 Hxd), 1.90–2.21 (m, 6H, g-CH2 Pro, b-
CH2 Pro, b-CH2 Met), 2.09 (s, 3H, CH3 Met), 2.38–2.64 (m, 2H, g-CH2
Met), 3.02(dd, 1H, J=13.7, 9.1 Hz, b-CH2 Cys), 3.23 (dd, 1H, J=13.6,
5.0 Hz, b-CH2 Cys), 3.30–3.45 (m, 2H, e-CH2 Pro), 3.75 (dd, 1H, J=10.9,
5.9 Hz, b-CH2 Ser), 3.85 (dd, 1H, J=10.9, 5.4 Hz, b-CH2 Ser), 3.94 (m,
2H, a-CH2 Gly), 4.31 (dd, 1H, J=8.4, 6.3 Hz, a-CH Pro), 4.40 (t, 1H,
J=5.7 Hz, a-CH Met), 4.56 (dd, 1H, J=9.0, 4.9 Hz, a-CH Ser), 4.62(d,
2H, J=6.7 Hz, CH2 Hxd), 4.69 (dd, 1H, J=9.0, 5.1 Hz, a-CH Cys), 5.61
(ddd, 1H, J=13.2, 6.5, 6.5 Hz, CH=CHCH3), 5.73–5.81 (m, 1H, CH2CH=
CH), 6.06 (dd, 1H, J=15.0, 10.4 Hz, CH=CHCH3), 6.27 ppm (dd, 1H,
J=15.1, 10.3 Hz, CH2CH=CH); ESI-MS: m/z: calcd for C28H47N5O7S3:
662.3; found: 662.2 [M+H]+, 684.3 [M+Na]+; MALDI-TOF: m/z: 662.8
[M+H]+, 684.8 [M+Na]+, 700.8 [M+K]+.
H-Lys-Phe-Pro-Ile-Gly-Leu-Gly-Phe-Gly-OHxd (4g): Starting from 3g
(5.4 mg, 3.7 mmol) a colorless solid (3.5 mg, 3.4 mmol, 92%) was obtained.
[a]2D0 =ꢁ25.78 (c=0.2, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.82
(dd, 3H, J=6.3, 3.1 Hz, CH3 Ile), 0.87 (d, 3H, J=6.5 Hz, CH3 Ile), 0.90–
1.00 (m, 6H, 2”CH3 Leu), 1.17–1.72(m, 10H, CH Ile, d-CH2 Lys, g-CH2
2
Lys, b-CH2 Leu, b-CH2 Lys), 1.73 (d, 3H, J=6.6 Hz, CH3 Hxd), 1.83–2.04
(m, 5H, b-CH2 Pro, g-CH2 Pro, g-CH Leu), 2.04–2.13 (m, 1H, b-CH Ile),
2.89–3.04 (m, 4H, 2”1H b-CH2 Phe, e-CH2 Lys), 3.17–3.26 (m, 2H, 2”
1H b-CH2 Phe), 3.32–3.52 (m, 1H, e-CH2 Pro), 3.72–4.02 (m, 8H, e-CH2
Pro, 3”a-CH2 Gly), 4.10 (m, 1H, a-CH), 4.21–4.28 (m, 2H, 2”a-CH),
H-Lys-Leu-Gly-Phe-Ala-Gly-OHxd (4c): Starting from 3c (7 mg,
0.0063 mmol) a colorless solid (3.5 mg, 0.0052mmol, 83%) was obtained.
[a]2D0 =ꢁ7.28 (c=0.3, MeOH); 1H NMR (CD3OD, 400 MHz): d=0.95 (d,
3H, J=6.5 Hz, CH3 Leu), 0.97 (d, 3H, J=6.5 Hz, CH3 Leu), 1.38 (d, 3H,
J=7.2Hz, CH 3 Ala), 1.45–1.91 (m, 9H, 3”CH2 Lys, b-CH2 Leu, g-CH
Leu), 1.74 (d, 3H, J=6.4 Hz, CH3 Hxd), 2.90–3.20 (m, 4H, b-CH2 Phe,
4.49–4.56 (m, 2H, 2” a-CH), 4.62(d, 2H,
J=6.5 Hz, CH2 Hxd), 5.62
(ddd, 1H, J=13.2, 6.7, 6.7 Hz, CH=CHCH3), 5.71–5.82(m, 1H, CH CH=
2
CH), 6.06 (dd, 1H, J=14.9, 10.5 Hz, CH=CHCH3), 6.27 (dd, 1H, J=
14.6, 10.0 Hz, CH2CH=CH), 7.19–7.35 ppm (m, 10H, Ar Phe); ESI-MS:
m/z: calcd for C53H78N10O10: 1015.6; found: 1015.7 [M+H]+; MALDI-
TOF: m/z: 1015.9 [M+H]+, 1037.8 [M+Na]+, 1053.8 [M+K]+.
CH2 Lys), 3.88–3.97 (m, 4H, 2” a-CH2 Gly), 4.34–4.42(m, 2H, 2”
a-
CH), 4.58–4.61 (m, 2H, 2” a-CH), 4.62(d, 2H, J=6.2Hz, CH 2 Hxd),
5.62(ddd, 1H, J=13.6, 6.6, 6.6 Hz, CH=CHCH3), 5.72–5.81 (m, 1H,
CH2CH=CH), 6.06 (dd, 1H, J=15.0, 10.5 Hz, CH=CHCH3), 6.28 (dd,
1H, J=15.1, 10.3 Hz, CH2CH=CH), 7.18–7.29 ppm (m, 5H, Ar Phe);
ESI-MS: m/z: calcd for C34H53N7O7: 671.4; found 671.4 [M]+; MALDI-
TOF: m/z: 672.9 [M+H]+, 694.9 [M+Na]+, 710.9 [M+K]+.
H-Cys(StBu)-Gly-Pro-Ala-Gly-OHxd (4h): Starting from 3h (70 mg,
E
0.09 mmol) a colorless solid (18 mg, 0.05 mmol, 35%) was obtained.
1
[a]2D0 =ꢁ18.58 (c=0.2, MeOH); H NMR (CD3OD, 400 MHz): d=1.37 (s,
9H, tBu), 1.38 (d, 3H, J=7.3 Hz, CH3 Ala), 1.75 (d, 2H, J=6.7 Hz, CH3
Hxd), 1.86–2.38 (m, 4H, b-CH2, g-CH2 Pro), 3.07 (dd, 1H, J=8.5,
14.3 Hz, b-CH2 Cys), 3.26 (dd, 1H, J=5.2, 14.1 Hz, b-CH2 Cys), 3.52–3.74
(m, 2H, d-CH2 Pro), 3.89–4.01 (m, 2H, a-CH2 Gly), 4.05–4.17 (m, 3H, a-
H-Lys-Leu-Gly-Lys(Mtt)-Ala-Gly-OHxd (4d): Starting from 3d (21 mg,
A
15.5 mmol) a colorless solid (10.2mg, 11.2 mmol, 73%) was obtained.
1H NMR (CD3OD, 400 MHz): d=0.94 (d, 3H, J=6.4 Hz, CH3 Leu), 0.96
(d, 3H, J=6.4 Hz, CH3 Leu), 1.29–1.40 (m, 2H, g-CH2 Lys), 1.39 (d, 3H,
J=7.2Hz, CH 3 Ala), 1.44–1.55 (m, 2H, g-CH2 Lys), 1.58–1.93 (m, 11H,
2”d-CH2 Lys, b-CH2 Leuk, 2” b-CH2 Lys, g-CH Leu), 1.74 (d, 3H, J=
6.6 Hz, CH3 Hxd), 2.38 (s, 3H, CH3 Mtt), 2.85–2.99 (m, 4H, 2”e-CH2
Lys), 3.77–3.96 (m, 5H, 2” a-CH2 Gly, a-CH), 4.26–4.43 (m, 3H, 3”a-
CH), 4.58 (d, 2H, J=6.6 Hz, CH2 Hxd), 5.59 (ddd, 1H, J=13.3, 6.6,
6.6 Hz, CH=CHCH3), 5.71–5.80 (m, 1H, CH2CH=CH), 6.05 (dd, 1H, J=
CH2 Gly, a-CH Cys), 4.36–4.46 (m, 2H, 2” a-CH), 4.62(d, 2H,
J=
6.6 Hz, CH2 Hxd), 5.61 (ddd, 1H, J=13.8, 7.1, 7.1 Hz, CH=CHCH3),
5.72–5.81 (m, 1H, CH2CH=CH), 6.06 (dd, 1H, J=15.1, 10.5 Hz, CH=
CHCH3), 6.28 (dd, 1H, J=15.2, 10.4 Hz, CH2CH=CH); ESI-MS: m/z:
calcd for C25H41N5O6S2: 572.3; found: 572.3 [M+H]+, 594.3 [M+Na]+;
FAB-HRMS: m/z: 571.2521 [M]+.
H-Lys-Cys-Gly-Val-Phe-Gly-OHxd (5): Peptide 4d (4.2mg, 5.4 mmol)
was dissolved in a degassed solution (800 mL) of 0.1m ammonium bicar-
Chem. Eur. J. 2006, 12, 6095 – 6109
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6105

H. Waldmann et al.
bonate/DMF 5:3 under an argon atmosphere. DTT (12 mL, 0.14 mmol)
dissolved in degassed DMF (12 mL) was added to the peptide solution
and the mixture was stirred under argon at room temperature for 2.5 h.
The crude product was dried by lyophilization, redissolved in DMF, fil-
tered and purified by reversed-phase HPLC, to afford a colorless solid
(2.7 mg, 3.9 mmol, 72%); 1H NMR (CD3OD, 400 MHz): d=0.73 (d, 3H,
J=6.8 Hz, CH3 Val), 0.82(d, 3H, J=6.8 Hz, CH3 Val), 1.28–1.33 (m, 1H,
SH), 1.47–1.54 (m, 2H, g-CH2 Lys), 1.63–1.72(m, 2H, d-CH2 Lys), 1.74
(d, 3H, J=6.6 Hz, CH3 Hxd), 1.86–1.92(m, 2H, b-CH2 Lys), 2.00–2.07
(m, 1H, b-CH Val), 2.81–3.03 (m, 4H, e-CH2 Lys, b-CH2 Phe), 3.20 (dt,
2H, J=8.8, 5.0 Hz, b-CH2 Cys), 3.77 (d, 1H, J=16.1 Hz, a-CH Val), 3.94
(s, 2H, a-CH2 Gly), 3.98 (t, 1H, J=6.4 Hz, a-CH Lys), 4.07 (t, 2H, J=
7.11 (d, 1H, J=2.3 Hz, Ar Trp), 7.30 (d, 1H, J=8.0 Hz, Ar Trp), 7.56 (d,
1H, J=7.8 Hz, Ar Trp), 7.86 (d, 1H, J=8.9 Hz, CONH), 8.02(d, 1H, J=
8.0 Hz, CONH), 8.10–8.12(m, 2H, 2ꢁCONH), 8.33 (d, 1H,
J=7.8 Hz,
CONH), 10.76 ppm (d, 1H, J=2.0 Hz, NH Trp); ESI-MS: m/z: calcd for
C33H41N7O12: 728.3; found: 728.1 [M+H]+; MALDI-TOF: m/z: 750.8
[M+Na]+, 766.8 [M+K]+.
N-Maleimido-Gly-Ala-Lys-Thr-Ser-Ala-Glu-Ser-Tyr-Ser-Gly-OH
(6d):
Starting from Wang resin (285 mg) loaded with Fmoc-glycine
(0.29 mmol), a colorless solid (196 mg, 0.17 mmol, 59%) was obtained.
[a]2D0 =ꢁ7.78 (c=0.3, DMF); 1H NMR (D2O, 400 MHz): d=1.05 (d, 3H,
J=6.4 Hz, CH3 Thr), 1.20–1.37 (m, 2H, g-CH2 Lys), 1.26 (d, 6H, J=
7.2Hz, 2”CH Ala), 1.47–1.57 (m, 2H, d-CH2 Lys), 1.58–1.78 (m, 2H, b-
3
6.4 Hz, a-CH2 Gly), 4.53 (t, 2H, a-CH), 4.62(d, 2H,
J=6.6 Hz, CH2
CH2 Lys), 1.79–1.97 (m, 2H, b-CH2 Glu), 2.31 (t, 2H, J=7.5 Hz, g-CH2
Glu), 2.82–2.92 (m, 4H, e-CH2 Lys, b-CH2 Tyr), 3.63–3.79 (m, 6H, 3”b-
CH2 Ser), 3.83 (s, 2H, a-CH2 Gly), 4.10–4.33 (m, 11H, 8”a-CH, a-CH2,
b-CH Thr), 4.50 (t, 1H, J=7.5 Hz, a-CH), 6.67 (d, 2H, J=8.5 Hz, Ar
Tyr), 6.80 (s, 2H, CH=CH), 7.00 ppm (d, 2H, J=8.5 Hz, Ar Tyr); ESI-
MS: m/z: calcd for C47H68N12O21: 1137.5; found: 1137.5 [M+H]+;
MALDI-TOF: m/z: 1137.7 [M+H]+, 1159.7 [M+Na]+, 1175.6 [M+K]+;
FAB-LRMS: m/z: 1136.45 [M]+.
Hxd), 4.66 (t, 2H, J=5.0 Hz, a-CH), 5.58–5.65 (m, 1H, CH=CHCH3),
5.72–5.81 (m, 1H, CH2CH=CH), 6.03–6.10 (ddd, 1H, J=15.0, 10.4 Hz,
CH=CHCH3), 6.25–6.32 (dd, 1H, J=15.1, 10.5 Hz, CH2CH=CH), 7.18–
7.30 ppm (m, 5H, Ar Phe); MALDI-TOF: m/z: calcd for C33H51N7O7S:
690.4; found: 691.0 [M+H]+, 713.0 [M+Na]+, 729.0 [M+K]+.
General procedure for the solid-phase synthesis of the maleimido-pep-
tides 6 using Wang resin: Wang resin (Novabiochem) was loaded with
Fmoc-Gly-OH using 4 equiv amino acid, 4 equiv DIC and 0.1 equiv
DMAP in DMF (overnight, quantitative loading by UV-Fmoc determina-
tion). After peptide chain assembly was complete (SPPS), the peptidyl
resin was treated with TFA containing 2.5% TIS and 2.5% H2O as scav-
engers for 2–3 h. After partial evaporation of the resulting solution, the
crude peptide was precipitated by adding diethyl ether, filtered and
washed with diethyl ether. Finally the products were redissolved in a mix-
ture of MeOH/water and lyophilized.
N-Maleimido-bAla-Ser-Lys-Thr-Lys(dansyl)-Gly-OH (6e): Starting from
E
Wang resin (147 mg) loaded with Fmoc-glycine (0.13 mmol) a colorless
solid (67 mg, 0.074 mmol, 57%) was obtained. [a]2D0 =ꢁ19.08 (c=0.4,
MeOH); 1H NMR (CD3OD, 400 MHz): d=1.16 (d, 3H, J=6.4 Hz, CH3
Thr), 1.22–1.43 (m, 4H, 2”g-CH2 Lys), 1.47–1.73 (m, 6H, 2” a-CH2 Lys,
b-CH2 Lys), 1.73–1.81 (m, 1H, b-CH2 Lys), 1.94–2.03 (m, 1H, b-CH2
Lys), 2.54 (t, 2H, J=6.7 Hz, CH2 bAla), 2.84 (t, 2H, J=6.3 Hz, e-CH2
Lys), 2.94–3.00 (m, 8H, e-CH2 Lys, 2” CH 3 Dan), 3.68–3.83 (m, 4H, CH2
bAla, b-CH2 Ser), 3.84–3.93 (m, 2H,a-CH2 Gly), 4.13–4.19 (m, 1H, b-CH
Thr), 4.25 (dd, 1H, J=9.1, 4.8 Hz, a-CH), 4.29–4.34 (m, 2H, 2”a-CH),
4.43 (dd, 1H, J=9.7, 4.7 Hz, a-CH), 6.79 (s, 2H, CH=CH), 7.39 (d, 1H,
J=7.6 Hz, Ar Dan), 7.59–7.64 (m, 2H, Ar Dan), 8.20 (d, 1H, J=7.3 Hz,
Ar Dan), 8.42(d, 1H, J=8.7 Hz, Ar Dan), 8.53 ppm (d, 1H, J=8.6 Hz,
Ar Dan); ESI-MS: m/z: calcd for C40H57N9O13S: 904.4; found: 904.7
[M+H]+; MALDI-TOF: m/z: 904.9 [M+H]+, 926.9 [M+Na]+, 942.9
[M+K]+; FAB-HRMS: m/z: 904.3904 [M+H]+.
N-Maleimido-Gly-Tyr-Thr-Gly-OH (6a): Starting from Wang resin
(301 mg) loaded with Fmoc-glycine (0.31 mmol) a colorless solid (91 mg,
0.19 mmol, 62%) was obtained. [a]2D0 =+16.28 (c=0.4, DMF); 1H NMR
(D2O, 400 MHz): d=0.99 (d, 3H, J=6.4 Hz, CH3 Thr), 2.81–2.90 (m, 2H,
b-CH2 Tyr), 3.77 (s, 2H, a-CH2 Gly), 4.02–4.07 (m, 1H, a-CH Thr), 4.10
(d, 2H, J=2.8 Hz, a-CH2 Gly), 4.17 (d, 1H, J=4.0 Hz, b-CH Thr), 4.50
(t, 1H, J=7.8 Hz, a-CH Tyr), 6.66 (d, 2H, J=8.4 Hz, Ar Tyr), 6.75 (s,
2H, CH=CH), 6.97 ppm (d, 2H, J=8.4 Hz, Ar Tyr); 13C NMR (D2O,
100 MHz): d=18.8, 36.4, 39.9, 41.4, 55.7, 58.9, 67.2, 115.7, 127.8, 130.7,
134.7, 154.5, 169.0, 171.7, 171.9, 172.7, 173.2 ppm; ESI-MS: m/z: calcd for
C21H24N4O9: 477.1; found: 477.0 [M+H]+, 499.0 [M+Na]+; FAB-HRMS:
m/z: 499.1458 [M+Na]+.
Fmoc-Lys
N
N
treated with TFA/DCM 1:1 (10 mL) for 80 min at room temperature.
Excess of TFA was removed by coevaporation with toluene and the pep-
tide was dried under reduced pressure (colorless oil, Fmoc-Lys-
OH·TFA). The side-chain deprotected peptide was dissolved in MeOH/
N-Maleimido-Gly-Thr-Gln-Phe-His-Gly-OH (6b): Starting from Wang
resin (276 mg) loaded with Fmoc-glycine (0.32 mmol), a colorless solid
(140 mg, 0.19 mmol, 60%) was obtained. [a]2D0 =ꢁ20.28 (c=1.0, DMF);
1H NMR ([D6]DMSO, 400 MHz): d=1.00 (d, 3H, J=6.2Hz, CH 3 Thr),
1.61–1.68 (m, 1H, b-CH2 Gln), 1.80–1.88 (m, 1H, b-CH2 Gln), 1.99–2.11
(m, 2H, g-CH2 Gln), 2.78 (dd, 1H, J=13.6, 9.4 Hz, b-CH2), 2.83–3.01 (m,
2H, 2”1H b-CH2), 3.11 (dd, 1H, J=15.2, 5.4 Hz, b-CH2), 3.68–3.84 (m,
2H, a-CH2 Gly), 3.91–3.98 (m, 1H, b-CH Thr), 4.14–4.30 (m, 4H, a-CH2
Gly, 2” a-CH), 4.43 (dd, 1H, J=8.8, 5.4 Hz, a-CH), 4.62(dd, 1H, J=7.7,
6.0 Hz, a-CH), 6.82(brs, 1H, CH His), 7.08 (s, 2H, CH =CH), 7.16–7.26
(m, 5H, Ar Phe), 7.35 (brs, 1H, CH His), 8.02(d, 1H, J=7.6 Hz,
CONH), 8.07 (d, 1H, J=7.5 Hz, CONH), 8.14 (t, 1H, J=5.8 Hz,
CONH), 8.20 (d, 1H, J=8.1 Hz, CONH), 8.35 (d, 1H, J=8.5 Hz,
CONH), 8.95 ppm (s, 1H, NH); ESI-MS: m/z: calcd for C32H39N9O11:
726.3; found: 726.3 [M+H]+; MALDI-TOF: m/z: 726.9 [M+H]+, 748.9
[M+Na]+, 764.9 [M+K]+.
H2O 5:2(35 mL), followed by addition of NaHCO (270 mg, 3.2 mmol)
3
and dansyl chloride (432mg, 1.6 mmol). The reaction mixture was stirred
for 19 h at room temperature. The pH was adjusted to 2by adding HCl
1m and the product was extracted 3”with DCM, washed with brine,
dried over Na2SO4 and concentrated in vacuo. Purification was per-
formed by silica gel flash chromatography, eluting first with DCM, fol-
lowed by DCM/MeOH 10:1, affording (336 mg, 0.56 mol, 52%) a light
yellow oil (fluorescent). Rf =0.10 (DCM/MeOH 10:1); [a]2D0 =ꢁ5.58 (c=
0.4, CHCl3); 1H NMR (CD3OD, 400 MHz): d=1.07–1.34 (m, 4H, g-CH2,
d-CH2), 1.35–1.70 (m, 2H, b-CH2), 2.77–2.84 (m, 2H, e-CH2), 2.79 (s, 6H,
2”CH3 Dan), 3.95 (dd, 1H, J=4.5, 9.2Hz, a-CH), 4.20 (t, 1H, J=
6.7 Hz, CH Fmoc), 4.33 (d, 2H, J=6.0 Hz, CH2 Fmoc), 7.06 (d, 1H, J=
7.1 Hz, Ar Dan), 7.10–7.21 (m, 2H, Ar Dan), 7.27 (t, 2H, J=7.4 Hz, Ar
Fmoc), 7.40 (t, 2H, J=7.4 Hz, Ar Fmoc), 7.50 (d, 2H, J=7.0 Hz, Ar
Fmoc), 7.64 (d, 2H, J=7.5 Hz, Ar Fmoc), 8.17 (d, 1H, J=7.2Hz, Ar
Dan), 8.34 (d, 1H, J=8.7 Hz, Ar Dan), 8.51 ppm (d, 1H, J=8.5 Hz, Ar
Dan); 13C NMR (CD3OD, 100 MHz): d=22.4, 29.1, 31.8, 42.8, 45.6, 47.3,
54.0, 67.2, 115.5, 119.3, 120.0, 123.5, 125.4, 127.1, 127.8, 128.5, 129.6,
129.8, 130.0, 130.5, 135.1, 141.4, 143.9, 144.1, 152.0, 156.8, 174.8 ppm;
MALDI-TOF: m/z: calcd for C33H35N3O6S: 600.2; found: 600.4 [MꢁH]+,
624.4 [M+Na]+.
N-Maleimido-Gly-Ser-Glu-Trp-Ile-Gly-OH (6c): Starting from Wang
resin (350 mg) loaded with Fmoc-glycine (0.40 mmol) a colorless solid
(152mg, 0.21 mmol, 53%) was obtained. [ a]2D0 =ꢁ24.5 (c=0.7, DMF);
1H NMR ([D6]DMSO, 400 MHz): d=0.80 (t, 3H, J=7.4 Hz, CH3 Ile),
0.84 (d, 3H, J=6.8 Hz, CH3 Ile), 1.00–1.13 (m, 1H, CH2 Ile), 1.38–1.47
(m, 1H, CH2 Ile), 1.65–1.75 (m, 2H, b-CH2 Glu), 1.83–1.92(m, 1H, b-
CH Ile), 2.19 (t, 2H, J=8.0 Hz, g-CH2 Glu), 2.89 (dd, 1H, J=14.8,
8.6 Hz, b-CH2 Trp), 3.11 (dd, 1H, J=14.8, 5.4 Hz, b-CH2 Trp), 3.49–3.59
General procedure for the Diels–Alder ligation: Diene- and dienophile–
peptides were dissolved in water at room temperature to a concentration
of 10 mm. If required, methanol or DMF was added in minimum amounts
to support peptide solubilization (Table 3). The ligation was monitored
by HPLC analysis. After appropriate reaction time (20–48 h), the ligation
(m, 2H, b-CH2 Ser), 3.65–3.80 (m, 2H, a-CH2 Gly), 4.12(d, 2H,
J=
5.5 Hz, a-CH2 Gly), 4.17–4.28 (m, 2H, 2” a-CH), 4.34 (dd, 1H, J=7.7,
5.9 Hz, a-CH), 4.59 (dd, 1H, J=8.1, 5.7 Hz, a-CH), 6.96 (t, 1H, J=
8.0 Hz, Ar Trp), 7.04 (t, 1H, J=8.0 Hz, Ar Trp), 7.07 (s, 2H, CH=CH),
6106
www.chemeurj.org
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 6095 – 6109

Diels–Alder Ligation
FULL PAPER
product was directly purified by reversed phase HPLC and finally lyophi-
lized.
protection groups from lysine side chain took place during the reaction,
giving two cycloadducts 8d (with Mtt) and 8d’ (without Mtt). These com-
pounds were isolated by reversed-phase HPLC in 64% overall yield. 8d:
colorless solid (1.2mg, 0.73 mmol, 40%), compound with Mtt group.
1H NMR (CD3OD, 400 MHz): d=1.40 (d, 3H, CH3 Hxd), 2.38 (s, 3H,
Mtt), 5.67–5.75 (m, 2H, CH=CH), 7.1–7.5 ppm (Mtt); MALDI-TOF: m/
z: calcd for C84H113N15O19: 1380.7; found: 1381.5 [(MꢁMtt)+H]+, 1403.4
[(MꢁMtt)+Na]+, 1419.4 [(MꢁMtt)+K]+, 257.3 [Mtt]+. 8d’: colorless
solid (0.6 mg, 0.43 mmol, 24%), compound without Mtt group. 1H NMR
H-Val-Ala-Gly-O-cyclo-N-Gly-Tyr-Thr-Gly-OH (8a): Stirring of Val-Ala-
Gly-OHxd (1; 11 mg, 0.033 mmol) and 6a (19 mg, 0.039 mmol) in H2O/
MeOH 10:3 (1 mL) for 20 h gave a colorless solid (23 mg, 0.029 mmol,
87%). [a]2D0 =+11.78 (c=1.0, DMF); 1H NMR (CD3OD, 400 MHz): d=
1.03 (d, 3H, J=6.8 Hz, CH3 Val), 1.06 (d, 3H, J=6.9 Hz, CH3 Val), 1.14
(d, 3H, J=6.4 Hz, CH3 Thr), 1.39 (d, 3H, J=7.2Hz, CH 3 Ala), 1.40 (d,
3H, J=7.0 Hz, CH3 cyclo), 2.14–2.24 (m, 1H, b-CH Val), 2.44–2.52 (m,
1H, H6), 2.65–2.72 (m, 1H, H3), 2.88 (dd, 1H, J=13.7, 7.8 Hz, b-CH2
Tyr), 3.02(dd, 1H, J=13.9, 6.3 Hz, b-CH2 Tyr), 3.18 (dd, 1H, J=8.4,
7.2Hz, H 5), 3.39 (dd, 1H, J=8.5, 6.1 Hz, H4), 3.67 (d, 1H, J=5.6 Hz, a-
CH Val), 3.84–4.09 (m, 6H, 3”a-CH2 Gly), 4.13–4.20 (m, 1H, b-CH
Thr), 4.31 (t, 1H, J=3.5 Hz, a-CH), 4.47 (ddd, 1H, J=14.3, 7.1, 3.2Hz,
CH2 cyclo), 4.51–4.63 (m, 3H, CH2 cyclo, 2” a-CH), 5.71–5.79 (m, 2H,
CH=CH), 6.69 (d, 2H, J=7.4 Hz, Ar Tyr), 7.04 ppm (d, 2H, J=8.5 Hz,
([D6]DMSO, 400 MHz): d=1.2(d, 3H, CH Hxd), 5.02–5.75 ppm (m,
3
2H, CH=CH); MALDI-TOF: m/z: calcd for C64H97N15O19
found: 1381.3 [M+H]+, 1403.2[ M+Na]+, 1419.2[ M+K]+.
:
1380.7;
H-Pro-Cys
A
(8e):
Stirring of 4b (2.0 mg, 3.0 mmol) and 6a (3.5 mg, 7.3 mmol) in H2O/
MeOH 3:2(500 mL) for 24 h gave a colorless solid (1.1 mg, 0.97 mmol,
32%. [a]2D0 =ꢁ31.58 (c=0.1, MeOH); 1H NMR (CD3OD, 400 MHz): d=
1.14 (d, 3H, J=6.5 Hz, CH3 Thr), 1.35 (brs, 9H, tBu Cys), 1.40/1.41
(ratio 1:1, d, 3H, J=7.0 Hz, CH3 cyclo), 1.89–2.21 (m, 5H, g-CH2 Pro, b-
CH2 Pro, b-CH2 Met), 2.08 (s, 3H, CH3 Met), 2.42–2.65 (m, 4H, b-CH2
Pro, H6, g-CH2 Met), 2.66–2.72 (m, 1H, H3), 2.85–2.92 (m, 1H, b-CH2
Tyr), 2.97–3.05 (m, 2H, b-CH2 Tyr, b-CH2 Cys), 3.2–3.5 (m, b-CH2 Cys,
H5, H4), 3.56–4.19 (m, 11H, d-CH2 Pro, b-CH2 Ser, 3”a-CH2 Gly, b-CH
Thr), 4.29–4.45 (m, 3H, 3”a-CH), 4.53–4.66 (m, 4H, 2” a-CH, CH2
cyclo), 4.69–4.73 (m, 1H, a-CH), 5.68–5.80 (m, 2H, CH=CH), 6.68/6.69
(ratio 1:1, d, 2H, J=8.5 Hz, Ar Tyr), 7.03/7.04 ppm (ratio 1:1, d, 2H, J=
8.3 Hz, Ar Tyr); ESI-MS: m/z: calcd for C49H71N9O16S3: 1138.4; found:
1138.4 [M+H]+; MALDI-TOF: m/z: 139.1 [M+H]+, 1161.0 [M+Na]+,
1177.0 [M+K]+.
Ar Tyr); ESI-MS: m/z: calcd for C37H51N7O13
: 802.4; found: 802.4
[M+H]+; MALDI-TOF: m/z: 802.9 [M+H]+, 824.9 [M+Na]+, 840.9
[M+K]+.
H-Lys-Pro-Phe-Leu-Gly-O-cyclo-N-Gly-Tyr-Thr-Gly-OH (8b): Stirring
of 4a (10 mg, 0.015 mmol) and 6a (7 mg, 0.015 mmol) at room tempera-
ture in H2O/MeOH 4:1 (1 mL) for 24 h gave a colorless solid (10 mg,
0.009 mmol, 60%). [a]2D0 =ꢁ23.28 (c=0.3, MeOH); 1H NMR (CD3OD,
400 MHz): d=0.90 (d, 3H, J=6.0 Hz, CH3 Leu), 0.94 (d, 3H, J=6.0 Hz,
CH3 Leu), 1.14 (d, 3H, J=6.4 Hz, CH3 Thr), 1.39/1.40 (ratio 1:1, d, 3H,
J=7.3 Hz, CH3 cyclo), 1.51–1.74 (m, 7H, g-CH2 Lys, d-CH2 Lys, b-CH2
Leu, g-CH Leu), 1.87–2.09 (m, 5H, g-CH2 Pro, b-CH2 Lys, b-CH2 Pro),
2.16–2.26 (m, 1H, b-CH2 Pro), 2.45–2.51 (m, 1H, H6), 2.64–2.73 (m, 1H,
H3), 2.87 (dd, 1H, J=13.9, 7.8 Hz, b-CH2 Tyr), 2.92–3.15 (m, 5H, b-CH2
Tyr, e-CH2 Lys, b-CH2 Phe), 3.13/3.20 (ratio 1:1, dd, 1H, J=8.5, 7.2/8.4,
7.2Hz, H 5), 3.38/3.46 (ratio 1:1, dd, 1H, J=8.4, 6.2/8.5, 6.3 Hz, H4), 3.55–
3.64 (m, 1H, d-CH2 Pro), 3.67–3.74 (m, 1H, d-CH2 Pro), 3.83–4.11 (m,
6H, 3”a-CH2 Gly), 4.13–4.19 (m, 1H, b-CH Thr), 4.23–4.28 (m, 1H, a-
CH), 4.32(t, 1H, J=3.7 Hz, a-CH), 4.39–4.46 (m, 1H, CH2 cyclo), 4.48–
H-Lys-Phe-Pro-Ile-Gly-Leu-Phe-Gly-O-cyclo-N-Gly-Ala-Lys-Thr-Ser-
Ala-Glu-Ser-Tyr-Ser-Gly-OH (8 f): Stirring of 4 f (2.8 mg, 2.9 mmol) and
6d (3.3 mg, 2.9 mmol) in H2O (300 mL) for 48 h gave a colorless solid
(3.5 mg, 1.7 mmol, 69%). [a]2D0 =ꢁ22.98 (c=0.1, MeOH); 1H NMR
(CD3OD, 400 MHz): d=0.81–1.00 (m, 12H, 2”CH 3 Ile, 2”CH3 Leu),
1.19 (d, 3H, J=6.3 Hz, CH3 Thr), 1.19–1.77 (m, 22H, 2”b-/g-/d-CH2 Lys,
2”CH3 Ala, CH2 Ile, b-CH2 Leu), 1.43 (d, 3H, J=7.3 Hz, CH3 cyclo),
1.81–2.20 (m, 8H, b-CH2 Glu, b-CH2 Pro, g-CH2 Pro, g-CH Leu, b-CH
Ile), 2.41–2.46 (m, 2H, g-CH2 Glu), 2.47–2.53 (m, 1H, H6), 2.66–2.74 (m,
1H, H3), 2.89–3.26 (m, 11H, 2”e-CH2 Lys, b-CH2 Tyr, 2” b-CH2 Phe,
H5), 3.34–3.57 (m, 3H, H4, e-CH2 Pro), 3.71–4.00 (m, 14H, 3”b-CH2 Ser,
4”a-CH2 Gly), 4.06–4.68 (m, 15H, 13”a-CH, CH2 cyclo), 5.71–5.77 (m,
2H, CH=CH), 6.68 (d, 2H, J=8.4 Hz, Ar Tyr), 7.08 (d, 2H, J=8.5 Hz,
Ar Tyr), 7.17–7.34 ppm (m, 10H, Ar Phe); ESI-MS: m/z: calcd for
C98H143N21O30: 1048; found: 1048 [M+H]2+; MALDI-TOF: m/z: 2098
[M+H]+, 2 12 0M[ +Na]+, 2135 [M+K]+.
4.52(m, 1H, CH cyclo), 4.54–4.65 (m, 4H, 4”a-CH), 5.70–5.78 (m, 2H,
2
CH=CH), 6.69 (d, 2H, J=8.4 Hz, Ar Tyr), 7.03 (d, 2H, J=8.5 Hz, Ar
Tyr), 7.18–7.31 ppm (m, 5H, Ar Phe); ESI-MS: m/z: calcd for
C55H76N10O15: 1117.6; found: 1117.6 [M+H]+; MALDI-TOF: m/z: 1118.0
[M+H]+, 1140.0 [M+Na]+, 1156.0 [M+K]+.
H-Lys-Leu-Gly-Lys(Mtt)-Ala-Gly-O-cyclo-N-Gly-Thr-Gln-Phe-His-Gly-
G
OH (8c): Compounds 4d (2.0 mg, 2.2 mmol) and 6b (1.6 mg, 2.2 mmol)
were stirred in H2O/MeOH 10:1 (200 mL) for 24 h. Partial removal of
Mtt protection groups from lysine side chain took place during the reac-
tion, giving two cycloadducts 8c (with Mtt) and 8c’ (without Mtt). These
compounds were isolated by reversed-phase HPLC in 74% overall yield.
8c: colorless solid (1.3 mg, 0.80 mmol, 43%), compound with Mtt group.
1H NMR (CD3OD, 400 MHz): d=0.94 (d, 3H, J=6.7 Hz, CH3 Leu), 0.96
(d, 3H, xJ=6.7 Hz, CH3 Leu), 1.20 (d, 3H, J=6.2Hz, CH 3 Thr), 1.39 (d,
6H, J=7.1 Hz, CH3 Ala, CH3 cyclo), 1.3–2.3 (m, CH2 Lys, CH2 Gln,
CH2,CH Leu), 2.37 (s, 3H, CH3 Mtt), 2.44–2.51 (m, 1H, H6), 2.63–2.72
(m, 1H, H3), 2.9–3.4 (m, b-CH2 Phe, b-CH2 His, b-CH2 Lys, H5, H4), 3.7–
4.7 (m, 5H, a-CH2 Gly, a-CH, CH2 cyclo), 5.72–5.76 (m, 2H, CH=CH),
7.17–7.49 (m, 20H, Ar Phe, Ar Mtt, CH His), 8.73 ppm (brs, 1H, NH
His); MALDI-TOF: m/z: calcd for C83H111N17O18: 1378.7; found: 1379.1
[(MꢁMtt)+H]+, 1417.1 [(MꢁMtt)+K]+, 257.3 [Mtt]+. 8c’: colorless solid
(0.8 mg, 0.58 mmol, 31%), compound without Mtt group. 1H NMR
(CD3OD, 400 MHz): d=0.95 (d, 3H, J=6.6 Hz, CH3 Leu), 0.98 (d, 3H,
J=6.6 Hz, CH3 Leu), 1.20 (d, 3H, J=6.4 Hz, CH3 Thr), 1.40 (d, 6H, J=
7.5 Hz, CH3 Ala, CH3 cyclo), 1.4–2.3 (m, CH2 Lys, CH2 Gln, CH2,CH
Leu), 2.47–2.53 (m, 1H, H6), 2.67–2.74 (m, 1H, H3), 2.9–3.4 (m, b-CH2
Phe, b-CH2 His, b-CH2 Lys, H5, H4), 3.7–4.7 (m, a-CH2 Gly, a-CH, CH2
cyclo), 5.73–5.77 (m, 2H, CH=CH), 7.35 (brs, 1H, CH His), 7.18–7.29
(m, 5H, Ar Phe), 8.71 ppm (brs, 1H, NH His); MALDI-TOF: m/z: calcd
H-Lys-Phe-Pro-Ile-Gly-Leu-Gly-Phe-Gly-O-cyclo-N-Gly-Ala-Lys-Thr-
Ser-Ala-Glu-Ser-Tyr-Ser-Gly-OH (8g): Stirring of 4g (2.8 mg, 2.7 mmol)
and 6d (3.1 mg, 2.7 mmol) in H2O (270 mL) for 48 h gave a colorless solid
(3.2mg, 1.5 mmol, 67%). [a]2D0 =ꢁ22.28 (c=0.1, MeOH); 1H NMR
(CD3OD, 400 MHz): d=0.82–1.00 (m, 12H, 2”CH3 Ile, 2”CH3 Leu),
1.19 (d, 3H, J=6.3 Hz, CH3 Thr), 1.20–1.74 (m, 22H, 2”b-/g-/d-CH2 Lys,
2”CH3 Ala, CH2 Ile, b-CH2 Leu), 1.43 (d, 3H, J=7.3 Hz, CH3 cyclo),
1.83–2.19 (m, 8H, b-CH2 Glu, b-/g-CH2 Pro, g-CH Leu, b-CH Ile), 2.41–
2.47 (m, 2H, g-CH2 Glu), 2.47–2.53 (m, 1H, H6), 2.66–2.74 (m, 1H, H3),
2.89–3.26 (m, 11H, 2”e-CH2 Lys, b-CH2 Tyr, 2” b-CH2 Phe, H5), 3.32–
3.57 (m, 3H, H4, e-CH2 Pro), 3.70–4.02(m, 16H, 3” b-CH2 Ser, 5”a-CH2
Gly), 4.06–4.68 (m, 16H, 14”a-CH, CH2 cyclo), 5.73–5.77 (m, 2H, CH=
CH), 6.68 (d, 2H, J=8.6 Hz, Ar Tyr), 7.06–7.09 (m, 2H, Ar Tyr), 7.17–
7.33 ppm (m, 10H, Ar Phe); ESI-MS: m/z: calcd for C100H146N22O31:
1076; found: 1076 [M+H]2+; MALDI-TOF: m/z: 2155 [M+H]+, 2177
[M+Na]+, 2193 [M+K]+.
Pimeloyl succinimidyl hexadienyl ester (9): Pimeloyl diene ester 10
(160 mg, 0.66 mmol), N-hydroxysuccinimide (96 mg, 0.83 mmol) and
DMAP (8.6 mg, 0.07 mmol) were dissolved in dry THF (6 mL). Then
DIC (118 mL, 0.76 mmol) was added dropwise at room temperature and
the reaction mixture was allowed to react overnight. The solvent was re-
moved under reduced pressure and the urea was precipitated by adding
EtOAc/cyclohexane and separated by filtration. The crude product was
purified by silica gel flash chromatography cyclohexane/EtOAc 3:1 !
for C63H95N17O18
:
1378.7; found: 1379.5 [M+H]+, 1401.4 [M+Na]+,
1417.4 [M+K]+.
H-Lys-Leu-Gly-Lys
ACHTREUNG
OH (8d): Compounds 4d (2.0 mg, 2.2 mmol) and 6c (1.6 mg, 2.2 mmol)
were stirred in H2O/DMF 4:1 (200 mL) for 48 h. Partial removal of Mtt
Chem. Eur. J. 2006, 12, 6095 – 6109
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6107

H. Waldmann et al.
1:1, to give a colorless oil (184 mg, 0.55 mmol, 82%). Rf =0.31 (cyclohex-
ane/EtOAc 1:1); 1H NMR (CDCl3, 400 MHz): d=1.38–1.46 (m, 2H,
CH2), 1.60–1.68 (m, 2H, CH2), 1.69–1.77 (m, 2H, CH2), 1.73 (d, 3H, J=
6.6 Hz, CH3 Hxd), 2.30 (t, 2H, J=7.5 Hz, CH2), 2.58 (t, 2H, J=7.5 Hz,
CH2), 2.80 (brs, 4H, 2”CH2 NHS), 4.54 (d, 2H, J=6.6 Hz, CH2 Hxd),
5.59 (ddd, 1H, J=14.0, 6.6, 6.6 Hz, CH=CHCH3), 5.68–5.77 (m, 1H,
CH2CH=CH), 6.02(dd, 1H, J=15.0, 10.7 Hz, CH=CHCH3), 6.21 ppm
(dd, 1H, J=15.1, 10.5 Hz, CH2CH=CH); 13C NMR (CDCl3, 100 MHz):
d=18.3, 24.4, 24.5, 25.8, 28.4, 30.9, 34.1, 65.0, 123.9, 130.6, 131.4, 135.0,
168.7, 169.4, 173.4 ppm; ESI-MS: m/z: calcd for C17H23NaNO6: 360.1;
found: 360.1 [M+Na]+; FAB-HRMS: m/z: 360.1448 [M+Na]+.
499.1; found: 499.5 [M+H]+; MALDI-TOF: m/z: 499.5 [M+H]+, 521.5
[M+Na]+, 537.5 [M+K]+.
Diels–Alder ligation of the streptavidin–diene conjugate and fluorescent-
ly labeled dienophiles (14): Hexadiene-conjugate 11 at 3–5 mgmLꢁ1 con-
centration in water was incubated with 30-fold excess of maleimides 6e,
12 or 13 and kept at 258C for 24 h while shaking. After this time, the
excess dienophile was removed by passing the reaction mixture through a
spin gel filtration column (DyeEx columns from Qiagen). The ligated
protein was analyzed by SDS-PAGE and MALDI-TOF experiments. The
same procedure was performed by carrying out the ligation of 11 with
100-fold excess of 6e in 20 mm sodium phosphate buffer pH 5.5, 6.0, 6.5
or 7.0.
Pimeloyl hexadiene ester (10): A solution of DIC (619 mL, 4 mmol) in
THF (5 mL) was added dropwise over a 30-minute period at room tem-
perature to a solution of pimelic acid (3.2g, 20 mmol), trans,trans-2,4-
hexadien-1-ol (393 mg, 4 mmol) and DMAP (49 mg, 0.4 mmol) in THF
(30 mL). The reaction mixture was stirred overnight. The solvent was
then evaporated and the urea was removed by precipitation with EtOAc/
cyclohexane. The filtrate was concentrated under reduced pressure and
the product was purified by silica gel flash chromatography cyclohexane/
EtOAc 10:3, affording a colorless oil (401 mg, 1.7 mmol, 43%). Rf =0.16
(cHex/EtOAc 10:3); 1H NMR (CDCl3, 400 MHz): d=1.33–1.41 (m, 2H,
CH2), 1.60–1.68 (m, 4H, 2”CH2), 1.75 (d, 3H, J=6.6 Hz, CH3 Hxd), 2.32
(t, 2H, J=7.4 Hz, CH2), 2.35 (t, 2H, J=7.4 Hz, CH2), 4.56 (d, 2H, J=
6.6 Hz, CH2 Hxd), 5.61 (ddd, 1H, J=13.6, 6.4, 6.4 Hz, CH=CHCH3),
5.70–5.79 (m, 1H, CH2CH=CH), 6.04 (dd, 1H, J=15.1, 10.5 Hz, CH=
CHCH3), 6.24 ppm (dd, 1H, J=15.2, 10.4 Hz, CH2CH=CH); 13C NMR
(CDCl3, 100 MHz): d=18.3, 24.5, 24.8, 28.7, 34.0, 34.3, 65.1, 123.9, 130.6,
131.5, 135.1, 173.6, 179.9 ppm; ESI-MS: m/z: calcd for C13H20NaO4:
263.1; found: 263.1 [M+Na]+; MALDI-TOF: m/z: 263.2 [M+Na]+, 279.2
[M+K]+.
Ligation of the Rab7DC3 thioester and peptides 16 or 4h (17): An ali-
quot of a stock solution (100 mL) of Rab7DC3 thioester 15 (6.8 mgmLꢁ1
in buffer 25 mm sodium phosphate pH 7.5, 25 mm NaCl, 0.5m MESNA,
10 mm GDP, 2m m MgCl2, 30 nmol) was combined with ligation buffer
(13 mL; 25 mm sodium phosphate pH 7.5, 100 mm NaCl, 100 mm MESNA,
2% CHAPS, 50 mm GDP, 1 mm MgCl2) and 10 mL of a solution of peptide
16 or 4h (60 mm in methanol, 600 nmol) was added. The final concentra-
tions were 250 mm for Rab7 thioester and 5 mm for the peptide (ca. 20
equivalents). The ligation reaction mixture was incubated overnight at
168C with slight shaking. Small samples were removed for analysis by
ESI-MS and SDS-PAGE. The resulting solution of ligated protein 17 was
directly submitted to the next step without further purification.
Masking of cysteine residues with Ellmannꢀs reagent (18): An aliquot of
dienyl Rab7 17 (ca. 5.7 mgmLꢁ1) solution (85 mL) was mixed with 30 mm
DTNB (115 mL; in 60 mm sodium phosphate pH 8) at 258C for 4 h. Sub-
sequently the yellowish reaction solution was dialyzed against DA buffer
(5 mm sodium phosphate buffer pH 6.0, 20 mm NaCl, 0.2m m MgCl2 and
20 mm GDP). The final concentration was approximately 1.2–1.5 mgmLꢁ1
of a colorless solution. Small samples were removed for analysis by ESI-
MS and SDS-PAGE. The protein solution was shock frozen and stored at
ꢁ808C.
Diels–Alder ligation of Rab7 hexadienyl ester 30 and maleimide com-
pounds (19): In different scale experiments, 10–300 mL of Rab7 hexadien-
yl ester 18 solution in DA buffer at concentration of approximately
1 mgmLꢁ1 (ca. 40 mm protein) were combined with 30 to 100 equivalents
of maleimide compounds 6e or 12. The ligation mixture was incubated at
258C for 24 h. Under these conditions, the ligated protein usually precipi-
tated gradually during the reaction course. The reaction was quenched by
adding 200 mm DTT (50 equiv relative to the amount of dienophile
added). The deprotection of the cystein residues was visually noticed by
the development of a yellow color upon addition of DTT resulted from
the release of the TNB groups into solution. After 2h, the reaction mix-
ture was analyzed by SDS-PAGE and ESI-MS.
Preparation of the streptavidin-diene conjugate (11)
A solution of streptavidin (1.09 mg, 21 nmol) in water (450 mL) was com-
bined with of a freshly prepared 50 mm solution (2.5 mL) of the diene
cross-linker 9 in DMF (125 nmol) for 30 min at 258C. The reaction mix-
ture was transferred to a Microcon centrifugal filtration device (10 kDa
cut-off), diafiltered with four changes of water and concentrated to a
final volume protein concentration of 19 mgmLꢁ1
N-Dansyl-2-maleimido-ethylamine (12):
2-Maleimido-ethylamine[19]
.
(38 mg, 0.15 mmol), dansyl chloride (54 mg, 0.20 mmol) and DIPEA
(78 mL, 0.45 mmol) were dissolved in 2.0 mL dry DMF and the solution
was stirred at room temperature for 1 h. Acetic acid (120 mL) was added
and the solution was concentrated under high vacuum. The residue was
redissolved in DCM and the organic solution was washed with HCl 0.1m
(3”), brine (1”), dried over Na2SO4 and concentrated. The product was
purified by reversed-phase HPLC and freeze-dried to give a light yellow
solid (45 mg, 0.12mmol, 80%). 1H NMR (CD3OD, 400 MHz): d=3.09–
3.13 (m, 2H, CH2), 3.11 (s, 6H, N(CH3)2), 3.45 (t, 2H, J=5.7 Hz, CH2),
N
Purification of the Rab7 cycloadduct 19a: After incubation of Rab7 hex-
adienyl ester 18a with maleimide 6e, the reaction mixture was centri-
fuged and the supernatant was removed. The pellet was washed with
methanol (2”) to remove excess dienophile and then redissolved in de-
naturating buffer (100 mm Tris-HCl pH 8.0, 6m guanidinium-HCl, 100 mm
6.45 (s, 2H, CH=CH maleoyl), 7.56 (d, 1H, J=7.7 Hz, Ar), 7.62–7.68 (m,
2H, Ar), 8.19 (d, 1H, J=7.3 Hz, Ar), 8.44 (d, 1H, J=8.6 Hz, Ar),
8.48 ppm (d, 1H, J=8.6 Hz, Ar); 13C NMR (CD3OD, 100 MHz): d=37.2
(CH2), 40.3 (CH2), 45.3 (N(CH3)2), 116.8 (Ar), 122.5 (Ar), 124.5 (Ar),
U
127.8 (Ar), 128.1 (Ar), 128.5 (Ar), 129.4 (Ar), 129.5 (Ar), 133.5 (CH=
CH), 170.9 ppm (C=O); MALDI-TOF: m/z: calcd for C18H19N3O4S:
374.1; found: 374.5 [M+H]+, 396.5 [M+Na]+, 412.5 [M+K]+; FAB-
HRMS: m/z: 373.1098 [M]+.
DTE, 1% CHAPS and 1 mm EDTA) to
a concentration of about
1 mgmLꢁ1 (the solution became yellowish because of the TNB group re-
lease) and incubated overnight at 48C. The protein was refolded by dilut-
ing it 25-fold dropwise with folding buffer (100 mm HEPES pH 7.5, 5 mm
DTE, 2m m MgCl2, 100 mm GDP, 1% CHAPS) and incubated at room
temperature for 3 h with slight stirring. The folded protein was submitted
to two different procedures respectively: 1) it was dialyzed against buffer
2 5 mm HEPES pH 7.5, 40 mm NaCl, 3 mm DTE, 2m m MgCl2 and 20 mm
GDP, concentrated by ultracentrifugation (Microcon 10 KDa cut-off) and
stored at ꢁ808C for subsequently use in spectrofluorometric assays; or 2)
an equimolar amount of the REP-1 protein was added and the solution
was incubated overnight at 48C, and the resulting complex was dialyzed
against 25 mm HEPES pH 7.5, 40 mm NaCl, 3 mm DTE, 2m m MgCl2 and
20 mm GDP, concentrated by ultracentrifugation (Amicon 10 KDa cut-
off) and stored at ꢁ808C.
N-Fluoresceinoyl-2-maleimido-ethylamine (13): 2-Maleimido-ethyla-
mine[19] (9 mg, 0.035 mmol), fluoresceine succinimidyl ester (15 mg,
0.028 mmol) and DIPEA (16 mL, 0.106 mmol) were dissolved in dry
DMF (1.0 mL) and the solution was stirred at room temperature for
2.5 h. Acetic acid (15 mL) was added and the product was directly puri-
fied by reversed-phase HPLC, affording
a yellow solid (6.5 mg,
0.013 mmol, 46%). 1H NMR (CD3OD, 400 MHz): d (I/II ratio 1:0.65) =
3.49/3.61 (t, 2H, J=6.1 Hz, CH2), 3.67/3.78 (t, 2H, J=5.0 Hz, CH2), 6.22–
6.67 (m, 2H, Xan), 6.71/6.82 (s, 2H, CH =CH maleoyl), 6.71–6.82(m, 2H,
Xan), 6.78–6.80 (m, 2H, Xan), 7.31 (d, 1H, J=8.0 Hz, Ar [II]), 7.54 (s,
1H, Ar [I]), 8.03 (d, 1H, J=8.0 Hz, Ar [I]), 8.11 (d, 1H, J=8.0 Hz, Ar
[I+II]), 8.34 ppm (s, 1H, Ar [II]); ESI-MS: m/z: calcd for C27H18N2O4:
6108
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ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 6095 – 6109

Diels–Alder Ligation
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Received: February 2, 2006
Published online: June 280, 2006
Chem. Eur. J. 2006, 12, 6095 – 6109
ꢁ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6109