Alternative solid-phase strategies for the efficient labelling of peptides with (bathophenanthroline)ruthenium(II) complexes

Article abstract of DOI:10.1002/hlca.200800409

To increase the flexibility for the insertion of highly sensitive and robust [Ru^II(bathophenanthroline)] complexes into peptides, we have evaluated three different solid-phase strategies (bathophenanthroline = 4,7-diphenyl-1,10-phenanthroline).

Full text of DOI:10.1002/hlca.200800409

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Helvetica Chimica Acta – Vol. 92 (2009)
Alternative Solid-Phase Strategies for the Efficient Labelling of Peptides with
(Bathophenanthroline)ruthenium(II) Complexes
by Rolf A. Kramer and Willi Bannwarth*
Institut fꢀr Organische Chemie und Biochemie, Albert-Ludwigs-Universitꢁt Freiburg, Albertstr. 21,
D-79104 Freiburg (phone: þ 497612036073; fax: þ 497612038705; e-mail:
willi.bannwarth@organik.chemie.uni-freiburg.de)
To increase the flexibility for the insertion of highly sensitive and robust [RuII(bathophenanthro-
line)] complexes into peptides, we have evaluated three different solid-phase strategies (batho-
phenanthroline ¼ 4,7-diphenyl-1,10-phenanthroline). Among these, insertion of the Ru-complex-modi-
fied lysine building block 9 turned out to be the method of choice (Scheme 5).
Introduction. – Fluorescence labelling of peptides is still an area of high interest.
Chelate complexes of the lanthanides EuIII and TbIII are the most prominent label
molecules for this purpose [1]. Their main advantage is represented by their strong
fluorescence and excited-state lifetimes up to milliseconds which allow for time-
resolved measurements virtually free of background.
For some time, our focus has been on ruthenium(II) charge-transfer complexes as
alternative label entities. They combine high thermodynamic stability with chemical
inertness and excited-state lifetimes in the microsecond range also allowing for highly
sensitive time-resolved measurements [2].
Meanwhile, we have applied these complexes in combination with suitable donor
and acceptor chromophores to robust fluorescence-resonance-energy-transfer (FRET)
systems either in peptides or in synthetic DNA fragments [3]. The dye preferably
applied to the labelling procedure is the [RuII(bathophenanthroline)] complex 1
depicted in Fig. 1 (bathophenanthroline ¼ 4,7-diphenyl-1,10-phenanthroline). The
ligand of complex 1 carries sulfonate groups to mediate solubility in aqueous systems
and one carboxy function which allows for attachments to amino functions of
biomolecules via stable amide bonds. Recently, we were able to improve the synthesis
of 1 substantially, and even a continuous large-scale preparation in a microreactor is
now possible [4].
A severe drawback of our attachment procedures of Ru-complex 1 to peptides has
been so far the restriction to N-terminal labelling1)2). To overcome this disadvantage
and to extend the scope of application, allowing to place the Ru-complex at will into
synthetic peptide sequences, we have contemplated three different solid-phase
approaches, all of which were evaluated for labelling of the e-amino group of l-lysine
1
)
)
For incorporation of EuIII complexes into peptides, see [5].
For reviews on the preparation of peptide – metal complex conjugates, see [6].
2
ꢂ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

Helvetica Chimica Acta – Vol. 92 (2009)
1047
Fig. 1. [RuII(bathophenanthroline)] complex 1 and model peptide 2
in the model peptide HꢀPheꢀLysꢀAspꢀHisꢀGlyꢀNH2 (2) (Fig. 1). Compared to
post-synthetic labelling procedures in solution, this would avoid final purification steps
to remove excesses of unreacted label.
Results and Discussion. – In the first approach outlined in Scheme 1, the peptide
was assembled employing the Fmoc/tBu strategy with the exception of l-lysine which
was protected at the e-amino function with the Dde protecting group developed by
Bycroft and co-workers [7] (Fmoc ¼ (3H-fluoren-9-ylmethoxy)carbonyl, Dde ¼ 1-(4,4-
dimethyl-2,6-dioxocyclohexylidene)ethyl. Insertion of the last amino acid in the Boc-
protected form and orthogonal removal of the Dde group of l-lysine with 2%
hydrazine was followed by quantitative acylation of the side-chain amino group of Lys
with Ru-complex 1 and TBTU as coupling reagent [8] (Boc ¼ (tert-butoxy)carbonyl,
TBTU ¼ 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate).
Scheme 1. Coupling of the Ru-Complex 1 to the e-Amino Group of l-Lysine
i) 2% Hydrazine in DMF, 4 ꢁ 3 min. ii) Ru-Complex 1, TBTU, iPr2EtN, DMF 15 h. iii) CF3COOH/H2O/
iPr3SiH 95 :2.5 :2.5; 2 h.

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Helvetica Chimica Acta – Vol. 92 (2009)
Cleavage of the protecting groups and removal from the support with CF3COOH
yielded the desired labelled peptide 3. Unfortunately, Ru-complex 1 revealed a high
tendency to stick to the polymer matrix so that part of it was released only during this
final CF3COOH treatment and appeared as impurity in the desired labelled peptide 3
making an additional purification step necessary (Fig. 2). The quantitative acylation
was confirmed by the absence of peptide 2 in the HPLC trace (tR 19.8 min).
Fig. 2. HPLC of crude peptide 3
The second approach was based on the introduction of a bathophenanthroline-
modified l-lysine, 5, during synthesis of the peptide. After assembly of the peptide
chain, the bathophenanthroline entity should be turned on the solid support into the
Ru-complex-labelled peptide 3. The bathophenanthroline-modified l-lysine 5 was thus
prepared from bathophenanthroline-derived ligand 4 and a-Fmoc-protected l-lysine
according to Scheme 2. The carboxy function of ligand 4 had to be activated with
HATU to reach a decent acylation rate [9] (HATU ¼ 2-(1H-7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate). The desired building block 5 was
obtained in a yield of 48% after chromatography (silica gel) during which the
bathophenanthroline entity severely hampered the purification procedure. The
envisaged transfer of the solid-phase-attached bathophenanthroline-labelled peptide
6 into the aspired peptide 3 should then be performed by reaction with Ru-complex 7a
or 7b according to Scheme 3. Initial experiments revealed that the exchange of the
chloride ligands of 7a by reaction with bathophenanthroline ligands proceeded very

Helvetica Chimica Acta – Vol. 92 (2009)
1049
slowly and required high reaction temperatures not compatible with sensitive
biomolecules. To overcome this limitation, we replaced in complex 7a the chloride
ions by the much weaker coordinating triflate ions (TfOꢀ)3) (! 7b). This was easily
achieved by treating complex 7a with silver triflate and filtering off the precipitated
AgCl.
Scheme 2. Synthesis of Ligand Building Block 5
i) HATU, iPr2EtN, DMF, 15 min. ii) Fmoc-Lys-OH, 15 h.
Scheme 3. Complexation of Ligand Peptide 6 with Two Different Ru-Complexes, 7a and 7b, on Solid
Support to Yield Peptide 3, after Final Deprotection and Cleavage from the Solid Support
We have so far no proof that the TfOꢀ (CF3SOꢀ3 ) is really coordinated or whether a solvent
molecule (MeOH) takes its place instead.
3
)

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A test reaction with peptide 8 in solution (Scheme 4) revealed indeed a much faster
ligand-exchange reaction when 7b instead of 7a was applied for the transfer into the
Ru-complex-labelled peptide 3. The pertinent kinetic data are depicted in Fig. 3.
Scheme 4. Complexation of Ligand Peptide 8 with Two Different Ru-Complexes 7a and 7b in Solution to
Yield Peptide 3
Fig. 3. Kinetics for the formation of labelled peptide 3 after reaction of the bathophenanthroline-labelled
peptide 8 with the Ru complexes 7a or 7b in solution

Helvetica Chimica Acta – Vol. 92 (2009)
1051
After these positive results, the solid-phase-bound peptide 6 was treated with an
excess of Ru-complex 7b at 608 for 24 h. After removal of the excess of 7b by intensive
washing steps, the cleavage of the protecting groups and the removal from the support
with CF3COOH yielded crude peptide 3 (Fig. 4) without any trace of complex 7b or
peptide 8. This indicated that the tendency to stick to the polymer matrix is much higher
for Ru-complex 1 than that of the complex 7b.
Fig. 4. HPLC of crude peptide 3 after reaction of Ru-complex 7b with the solid-phase-bound peptide 6
The ultimate strategy would be the application of a Ru-complex-labelled Fmoc-l-
lysine building block during the assembly of the peptide on solid support. This would be
compatible with automated peptide synthesis and require no additional modification
procedures as in the previous two strategies. Tor and co-workers have recently reported
on the preparation of a cysteine building block of a Ru-complex bearing two 1,1’-
bis[pyridines] and phenanthroline as ligands [10]. Unfortunately, the reported strategy
cannot be applied to the bathophenanthroline complexes. Therefore, we envisaged the
synthesis of building block 9 for this purpose.
Initial attempts to prepare 9 via coupling of Ru-complex 1 to the free e-amino group
of Fmoc-Lys-OH failed due to difficulties in the separation of the Ru-complex 1 from
the desired product 9. Therefore, we prepared building block 9 in an alternative way
starting from the bathophenanthroline-modified l-lysine derivative 5 and the Ru-
complex 7b4) according to Scheme 5. Purification of the crude material by chromatog-
raphy (silica gel) was to no avail but prep. MPLC on reversed-phase silica gel (C18)
yielded the desired pure building block 9 in a yield of 79%. Next, peptide 3 was
prepared employing standard Fmoc-building blocks in combination with Ru-complex-
4
)
7a as well as 7b contained about 7% of [Ru(bpds)3] (see [4]).

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Helvetica Chimica Acta – Vol. 92 (2009)
modified building block 9 in the fourth position from the C-terminus. All couplings
were performed with TBTU as activator. Final deprotection and removal from the
support with CF3COOH yielded the peptide without any by-products (Fig. 5) obviating
additional purification steps and indicating at the same time impressively the high
Scheme 5. Preparation of the Ru-Complex-Labelled l-Lysine Building Block 9
Fig. 5. HPLC of crude peptide 3 obtained by the building-block approach with incorporation of 9

Helvetica Chimica Acta – Vol. 92 (2009)
1053
efficiency with which building block 9 had been incorporated. A slight excess of 1.2
equiv. of 9 had resulted in complete coupling.
Conclusions. – In this study, we evaluated three different strategies to increase the
flexibility for the insertion of [RuII(bathophenanthroline)] complexes into peptides,
which so far was limited to attachment at the N-terminus of peptides. All three methods
are taking advantage of solid-phase chemistry allowing to remove excesses of reagents
by washings. The first approach, based on an orthogonal protection of the e-amino
group of l-lysine with Dde yielded the desired labelled model peptide 3 together with
Ru-complex 1 so that an additional purification was necessary. The second strategy, in
which the complete Ru-complex was formed directly on the solid support from a
bathophenanthroline-modified l-lysine of the peptide and complex 7b yielded the
desired peptide 3 with high efficiency and high purity. Finally, we were able to prepare a
[RuII(bathophenanthroline)] complex – l-lysine building block 9 and were able to
insert it into the peptide sequence during standard cycles and with high efficacy. Due to
the high stability of the applied Ru-complexes towards CF3COOH, no modifications or
decompositions of the label were observed during deprotection. Since this last
approach is compatible with automated peptide synthesis and requires no additional
manipulations, it is recommended for the preparation of Ru-complex-labelled peptides.
Experimental Part
General. All reagents were purchased from commercial sources with the exception of compounds 1,
4, and 7a, which were synthesized according to our established procedures [3]. The Ru-complex 7a was
converted to 7b by dissolving 7a in MeOH followed by addition of the appropriate amount of AgOTf.
Amine-free DMF (Roth) was employed throughout Ru-complex and peptide synthesis. Peptide
syntheses were carried out on a 0.02 mmol scale by the Fmoc/tBu protocol [11] and Tentagel-S-RAM resin
(loading 0.24 mmol gꢀ1) with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU) as a coupling reagent [8]. l-Aspartic acid as tBu ester (Fmoc-Asp(OtBu)-OH) and l-histidine
as trityl (Fmoc-His(Trt)-OH) side-chain-protected amino acids were employed. CC ¼ column chroma-
tography. HPLC: Agilent-1100 system with a Source-5RPC-ST-4.6/150 column (Amersham Pharmacia
Biotech). MPLC: purification of the Ru-complex – amino acid 9 with a Bꢀchi MPLC system (fraction
collector C660, pump module C605, pump manager C615, and UV photometer C635); the C18 reversed-
phase material for MPLC was synthesized by a modified standard procedure according to [12]. NMR
Spectra: at 400 MHz (1H), and at 100.6 MHz (13C); chemical shifts d in ppm rel. to the respective solvent
signals, J in Hz. MS: Finnigan MAT-8200 (EI), TSQ-7000 (ESI); in m/z. HR-MS: Bruker microToF-Q
´
´
equipped with a Bruker Appollo source; by the Institut Federatif de Recherche 85 at the Louis Pasteur
University, Strasbourg.
(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-6-{{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-
4-yl)phenyl]pentyl}amino}hexanoic Acid ¼ N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-{1-oxo-5-[4-(7-
phenyl-1,10-phenanthrolin-4-yl)phenyl]pentyl}-l-lysine; 5). Under Ar, a mixture of 5-[4-(7-phenyl-
1,10-phenanthrolin-4-yl)phenyl]pentanoic acid (4; 1.00 g, 2.31 mmol, 1.0 equiv.), (2-1H-7-azabenzotria-
zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU; 0.88 g, 2.31 mmol, 1.0 equiv.), and
iPr2EtN (0.40 ml, 4.62 mmol, 2.0 equiv.) in DMF (20 ml) was stirred for 15 min at r.t. and then treated
with Fmoc-Lys-OH (0.85 g, 2.31 mmol, 1.0 equiv.). The resulting suspension was diluted with DMF
(40 ml) and stirred again for 15 h at r.t. (!clear soln.). After evaporation of the solvent, the residue was
suspended in H2O (100 ml) and the mixture acidified with 2m HCl to a pH of 2. The resulting precipitate
was filtered off, washed with H2O (100 ml), and then purified by CC (SiO2, CH2Cl2/MeOH/AcOH
98 :2 :0.1 ! 90 :10 :0.1). From the pure fractions, AcOH was removed by co-evaporation with hexane: 5

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Helvetica Chimica Acta – Vol. 92 (2009)
(0.88 g, 48%). Pale pink powder. 1H-NMR (400 MHz, CD3OD): 1.39 – 1.49 (m, NHCH2CH2CH2); 1.50 –
1.59 (m, CH2CHCOOH); 1.64 – 1.76 (m, C6H4ꢀCH2CH2CH2, NHCH2CH2); 2.21 – 2.27 (m, NHCOCH2);
2.69 – 2.75 (m, C6H4ꢀCH2); 3.20 (t, J ¼ 6.6, NHCH2); 4.04 (t, J ¼ 7.5, HꢀC(9) of Fmoc); 4.11 – 4.17 (m,
CHꢀCOOH); 4.18 – 4.23 (m, CH2 of Fmoc); 7.20 (dd, J ¼ 7.4, 4.6, HꢀC(2,7) of Fmoc); 7.27 (d, J ¼ 7.4,
HꢀC(4) of Fmoc); 7.29 (d, J ¼ 7.4, HꢀC(5) of Fmoc); 7.37 (d, J ¼ 8.1, HꢀC(3,5) of C6H4(CH2)4); 7.44 (d,
J ¼ 8.2, HꢀC(2,6) of C6H4(CH2)4); 7.51 – 7.58 (m, 7 arom. H); 7.64 (d, J ¼ 7.3, HꢀC(3,6) of Fmoc); 7.77
(d, J ¼ 4.9, HꢀC(3) of phen); 7.80 (d, J ¼ 4.8, HꢀC(8) of phen); 7.88 (d, J ¼ 9.5, HꢀC(1) of Fmoc); 7.92
(d, J ¼ 9.5, HꢀC(8) of Fmoc); 9.08 (d, J ¼ 4.9, HꢀC(2) of phen); 9.13 (d, J ¼ 4.8, HꢀC(9) of phen).
13C-NMR (100.6 MHz, CD3OD): 24.32; 26.60; 29.92; 31.88; 32.34; 36.26; 36.87; 39.99; 55.30; 67.87; 68.11;
120.82; 125.74; 125.80; 125.95; 126.17; 126.20; 128.09; 128.71; 130.06; 130.14; 130.45; 130.79; 130.89;
135.26; 137.93; 142.37; 143.60; 143.74; 145.02; 145.18; 145.39; 148.75; 149.39; 152.56; 153.21; 158.59;
175.98. EI-MS: 805 (10, [M þ Na]þ), 783 (100, [M þ H]þ). Anal. calc. for C50H46N4O5: C 76.70, H 5.92, N
7.16; found: C 76.56, H 5.83, N 7.22.
Sodium Hydrogen {(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-6-{{1-oxo-5-[4-(7-phenyl-
1,10-phenanthrolin-4-yl-kN1,kN10)phenyl]pentyl}amino}hexanoato}bis{(1,10-phenanthroline-4,7-diyl-
kN1,kN10)bis[benzenesulfonato](2ꢀ)}ruthenate(3ꢀ) Trifluoromethanesulfonate (4 :1:1:2) (¼ Sodium
Hydrogen {N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N6-{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-4-yl-
kN1,kN10)phenyl]pentyl}-l-lysinato}bis{(1,10-phenanthroline-4,7-diyl-kN1,kN10)bis[benzenesulfonato]-
(2ꢀ)}ruthenate(3ꢀ) 1,1,1-Trifluoromethanesulfonate (4 :1:1:2); 9 · 2 TfOꢀ). A soln. of the Ru-complex
7b (465.7 mg, 0.304 mmol, 2.1 equiv.) and ligand 5 (250.0 mg, 0.319, 2.1 equiv.) in DMF (15 ml) was
stirred at 608 for 24 h. The mixture was concentrated and purified by reversed-phase MPLC (C18, H2O/
MeCN/CF3COOH 85 :15 :0.1 ! 67:33 :0.1). Lyophylization of the combined product fractions gave 9 ·
2 TfOꢀ (0.522 g, 79%). Red powder. HR-ESI-MS: 932.1557 (C100H78F6N8O23RuS26ꢀ, [M ꢀ 4 H ꢀ
2 OTfꢀ]2ꢀ; calc. 932.1567).
Sodium Bis{(1,10-phenanthroline-4,7-diyl-kN1,kN10)bis[benzenesulfonato](2ꢀ)}{l-phenylalanyl-N6-
{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-4-yl-kN1,kN10)phenyl]pentyl}-l-lysyl-l-a-aspartyl-l-histidyl-
glycinamide}ruthenate(2ꢀ) Chloride (4 :1:2) (3 · 2 Clꢀ) by the Orthogonal Protecting Group Strategy.
Standard peptide synthesis was employed on a 0.02 mmol scale [11]. During peptide synthesis, l-lysine
was introduced as the protected Fmoc-Lys(Dde)-OH and the last amino acid, l-phenylalanine, was
introduced as Boc-Phe-OH. The Dde deprotection was accomplished via the addition of 2% (v/v)
hydrazine/DMF (3 ꢁ 3 ml) and shaking for 3 min. For the coupling of the Ru-complex 1 · 2 Clꢀ, a soln. of
1 · 2 Clꢀ (52.5 mg, 0.03 mmol, 1.5 equiv.) in DMF (2 ml), TBTU (9.6 mg, 0.03 mmol, 1.5 equiv.), and
iPr2EtN (27.4 ml, 0.16 mmol, 8.0 equiv.) were added to the resin, which was agitated for 15 h. Afterwards,
the solid support was filtered and washed alternately with DMF/iPrOH (5 – 3 ml), before the peptide was
deprotected and cleaved from the solid support by exposure to CF3COOH/H2O/iPr3SiH 95 :2.5 :2.5 for
2 h. The cleavage cocktail was treated with Et2O to precipitate peptide derivative 3 · 2 Clꢀ and Ru-
complex 1 · 2 Clꢀ. Isolation of 3 · 2 Clꢀ was finally achieved by prep. HPLC. ESI-MS: 1051 (100, [M ꢀ
2 Cl ꢀ 4 Na þ 4 H]2þ).
l-Phenylalanyl-N6-{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-4-yl)phenyl]pentyl}-l-lysyl-l-a-aspar-
tyl-l-histidylglycinamide (8). Standard peptide synthesis was employed on a 0.02 mmol scale. As fourth
amino acid, the ligand amino acid 5 (20.4 mg, 0.026 mmol, 1.3 equiv.) was introduced with TBTU
i
(10.9 mg, 0.026 mmol, 1.3 equiv.) and Pr2EtN (27.4 ml, 0.16 mmol, 8.0 equiv.) in DMF (2 ml). For this
amino acid, the coupling time was extended to 15 h. The ligand-containing peptide 8 was isolated as a
pale pink powder. ESI-MS: 1017 (100, [M þ H]þ).
Sodium Ruthenate(2ꢀ) Chloride (4 :1:2) 3 · 2 Clꢀ or Sodium Ruthenate(2ꢀ) Trifluoromethanesul-
fonate (4 :1:2) 3 · 2 TfOꢀ by Complexation of Peptide 8 with the Ru-Complex 7a or 7b in Solution. Two
Eppendorf tubes were filled in parallel with stock solns. of Ru-complex 7a or 7b (c ¼ 0.015m, 0.18 ml,
2.7 mmol, 1.5 equiv.), ligand peptide 8 (2.0 mg, 1.8 mmol, 1.0 equiv.), and DMF (0.8 ml). The two
Eppendorf tubes were agitated in a thermomixer at 608 and 600 rpm. The samples were analyzed by
HPLC to determine the complexation efficiency. Isolation of 3 · 2 Xꢀ (Xꢀ ¼ Clꢀ or TfOꢀ) was finally
achieved by prep. HPLC. ESI-MS: 1051 (100, [M ꢀ 2 X ꢀ 4 Na þ 4 H]2þ).
Sodium Bis{(1,10-phenanthroline-4,7-diyl-kN1,kN10)bis[benzenesulfonato](2ꢀ)}{l-phenylalanyl-N6-
{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-4-yl-kN1,kN10)phenyl]pentyl}-l-lysyl-l-a-aspartyl-l-histidyl-

Helvetica Chimica Acta – Vol. 92 (2009)
1055
glycinamide}ruthenate(2ꢀ) 1,1,1-Trifluoromethanesulfonate (4 :1:2) (3 · 2 TfOꢀ) by Complexation of
Peptide 6 with the Ru-Complex 7b on Solid Support. Peptide 6 was synthesized on a 0.02 mmol scale on
solid support. After coupling of Boc-Phe-OH as last amino acid, a stock soln. of Ru-complex 7b (c ¼
0.015m, 2.0 ml, 30 mmol, 1.5 equiv.) was added. The resin was agitated in a thermomixer at 608 and
600 rpm for 24 h. The solid support was filtered and washed alternately with DMF/iPrOH (5 ꢁ 3 ml),
before the peptide was deprotected and cleaved from the solid support by exposure to CF3COOH/H2O/
iPr3SiH 95 :2.5 :2.5 for 2 h. Addition of Et2O precipitated 3 · 2 TfOꢀ which was obtained in pure form
after lyophilization (8.2 mg, 19%). Red powder. ESI-MS: 1051 (100, [M ꢀ 2 TfO ꢀ 4 Na þ 4 H]2þ).
Sodium Ruthenate(2ꢀ) Trifluoromethanesulfonate (4 :1:2) 3 · 2 TfOꢀ from Amino Acid Derived Ru-
Complex 9 · 2 TfOꢀ. Standard peptide synthesis was employed on a 0.02 mmol scale but as fourth amino
acid, the amino acid derived Ru-complex 9 · 2 TfOꢀ (45.0 mg, 0.024 mmol, 1.2 equiv.) in DMF (2 ml),
i
TBTU (7.7 mg, 0.024 mmol, 1.2 equiv.), and Pr2EtN (27.4 ml, 0.16 mmol, 8.0 equiv.) were added to the
resin, which was agitated for 15 h. The solid support was filtered and washed alternately with DMF/
iPrOH (5 ꢁ 3 ml). Deprotection and cleavage from the solid support was performed by exposure to
CF3COOH/H2O/iPr3SiH 95 :2.5 :2.5 for 2 h. After precipitation with Et2O and lyophilization, 3 · 2 TfOꢀ
was obtained (15.4 mg, 35%). Red powder. ESI-MS: 1051 (100, [M ꢀ 2 TfO ꢀ 4 Na þ H]2þ).
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[12] H. Glatz, C. Blay, H. Engelhardt, W. Bannwarth, Chromatographia 2004, 59, 567.
Received November 12, 2008