Anhydrotetracycline-peptide conjugates as representatives for ligand-based transactivating systems

Article abstract of DOI:10.1016/j.bmc.2010.06.061

Bioconjugates of anhydrotetracycline and minimal activation sequences (VP1, VP2) derived from the Herpes simplex virus protein VP16 were synthesized. Different ligation strategies were applied and the resulting molecules tested in HeLa cells expressing the reverse transactivator rtTA-S3 for activity. The data clearly demonstrate that the atc-peptide conjugates are able to penetrate the cell membrane. Furthermore, binding to and induction of rtTA-S3 were detected. Structure-activity relationships indicated that the biological activity of the atc-peptide strongly depends on the specific linker used. The N-terminally linked oxime derivative 10 proved excellent activity when the increase of luciferace activity indicated a transcriptional activation substantially exceeding the inducing properties of anhydrotetracycline.

Full text of DOI:10.1016/j.bmc.2010.06.061

Bioorganic & Medicinal Chemistry 18 (2010) 6127–6133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anhydrotetracycline–peptide conjugates as representatives for ligand-based
transactivating systems
b
Susanne Lochner a, Juergen Einsiedel a, Gesa Schaefer b, Christian Berens b, , Wolfgang Hillen ,
*
Peter Gmeiner a,
*
a Department of Chemistry and Pharmacy, Emil Fischer Center, Chair of Medicinal Chemistry, Friedrich Alexander University, Schuhstraße 19, D-91052 Erlangen, Germany
b Department Biology, Chair of Microbiology, Friedrich Alexander University, Staudtstraße 5, D-91058 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioconjugates of anhydrotetracycline and minimal activation sequences (VP1, VP2) derived from the Her-
pes simplex virus protein VP16 were synthesized. Different ligation strategies were applied and the
resulting molecules tested in HeLa cells expressing the reverse transactivator rtTA-S3 for activity. The
data clearly demonstrate that the atc-peptide conjugates are able to penetrate the cell membrane. Fur-
thermore, binding to and induction of rtTA-S3 were detected. Structure–activity relationships indicated
that the biological activity of the atc-peptide strongly depends on the specific linker used. The N-termi-
nally linked oxime derivative 10 proved excellent activity when the increase of luciferace activity indi-
cated a transcriptional activation substantially exceeding the inducing properties of anhydrotetracycline.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 10 March 2010
Accepted 16 June 2010
Available online 22 June 2010
Keywords:
Anhydrotetracycline
Peptide conjugates
TetR
Tetracycline
Transcriptional transactivation
Bioconjugation
1. Introduction
cline derivative (Fig. 2). Such a synthetic platform could serve as
first step for establishing highly flexible and versatile regulatory
Regulatory systems based on bacterial Tet repressor (TetR) are
widely used to conditionally alter the expression of selected target
genes in both model organisms and cultured cell lines.1 Spatially
and temporally controlled inducible expression allows in-depth
analysis of an individual gene’s contribution to normal cell physi-
ology, development or disease.2 TetR-based transregulators re-
spond to tetracycline,3 anhydrotetracyline (Fig. 1) and synthetic
derivatives thereof,4,5 which penetrate biological membranes
non-specifically, including the placenta and the blood–brain bar-
rier.2a,6 Screening and selection of TetR mutants allowed to estab-
lish a regulatory system with a reversed phenotype (revTetR),
that—in contrast to TetR—binds DNA only in the presence of tetra-
cyclines.7 For application in eukaryotic systems, TetR-based trans-
activators are typically constructed as artificial transcription
factors by fusing an N-terminally located TetR or revTetR unit with
an acidic transcriptional transactivation domain,8 like the one de-
rived from the Herpes simplex virus transactivator protein VP16.
As an alternative to these transcription factor-based systems,
we aimed to create a ligand-based activating molecule by cova-
lently attaching an activating peptide to the TetR-binding tetracy-
systems that can deliver many different kinds of biological readout,
but only need to employ a single Tet-responsive protein. Short pep-
tidic sequences that recruit proteins active in transcription regula-
tion are widespread in proteins or are also easily selected by
standard phage display techniques.9 To realize this project, we
chose to conjugate anhydrotetracycline (atc), which has high rev-
TetR affinity, with the octapeptide DFDLDMLG (VP1), the minimal
activating sequence derived from VP16 (Fig. 2b).10
As an attempt to improve the peptide’s activating properties,
VP2,10 a 16-mer consisting of two VP1 units, should be attached
as well. Since the crystal structure of TetR/glycylcycline11 com-
plexes showed a tunnel-like cavity around position 9 of the tetra-
cycline core leading to the surface of the protein,11a we selected a
9-substituted atc-derivative for bioconjugation. 9-Aminoanhydro-
tetracycline12 (H2N-atc) was the precursor of choice, since we
deemed the primary amine function most promising to allow
direct introduction of protected peptides via their C-terminus or
N
CH3
R = H:
anhydrotetracycline (atc)
OH
NH2
R = H2N:
R
* Corresponding authors. Tel.: +49 9131 8528084; fax: +49 9131 8528082 (C.B.),
tel.: +49 9131 8529383; fax: +49 9131 8522585 (P.G.).
OH
9-aminoanthydrotetracycline (H2N-atc)
O
O
O
OH OH
E-mail addresses: cberens@biologie.uni-erlangen.de (C. Berens), peter.gmei-
ner@medchem.uni-erlangen.de (P. Gmeiner).
Figure 1. Anhydrotetracyclines.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.061

6128
S. Lochner et al. / Bioorg. Med. Chem. 18 (2010) 6127–6133
following acylation reactions. The PyBOP-mediated coupling to 9-
amino-atc was accelerated by microwave irradiation which over-
came the low reactivity of the ortho-substituted amine moiety. Fi-
nally, cleavage of the tBu-esters and the Boc-protecting group with
TFA/DCM and RP18-HPLC purification allowed to obtain the atc-
VP1 conjugate 4 and the VP2-conjugate 5, each as two HPLC-sepa-
rable isomers. In both cases, the major product displayed the high-
er retention time. Interestingly, solution of the purified compounds
in methanol resulted in a conversion of the major to the minor iso-
mer and vice versa. This was also observed upon re-investigating
the VP2-conjugate solution in pyridine-d6 after measuring the 1H
NMR spectrum. We excluded the formation of a 4-epi-derivative
which might have been formed during the acidic peptide deprotec-
tion procedure, because the resonance of H-4a displayed the
10.6 Hz coupling constant typical for a non-epimerized spacial ori-
entation of H-4 and H-4a (for comparison e.g., see atc-derivative 7).
A dynamic isomerization of an amino acid in the conjugated pep-
tide chain can also be precluded. In our opinion, a plausible expla-
nation is the co-existence of two slowly interconverting tautomers
of the vinylogous carboxylic acid formed by the positions 1–3 of
the tetracycline ring system.
The synthesis of the N-terminally linked peptide conjugates fol-
lowed the methodology of oxime14 or maleimide/thiol15 ligations.
Acylation of 9-amino-atc with commercially available 4-formyl-
benzoic acid or 3-maleimidopropionic acid gave the functionalized
atc-derivatives 6 and 7, respectively (Scheme 2).
Peptide synthesis was performed starting from Fmoc-protected
Rink-amide AM resin which was treated with piperidine solution
and subsequently acylated with HATU-activated N-Fmoc-11-ami-
no-3,6,9-trioxaundecanoic acid employing microwave irradiation
(Scheme 3). After capping unreacted amine functions with acetic
acid anhydride and subsequent deprotection of the Fmoc-function,
the amino acid building blocks were incorporated as described
above. At the N-terminus, either the hydroxylamine derivative
bis-Boc-amino-oxyacetic acid or the protected thiol derivative tri-
tylsulfamylpropionic acid was attached. Cleavage from the resin
was achieved with a mixture of TFA and scavengers to yield the
hydroxylamine functionalized peptide 8 and the thiol analogue 9.
Oxime formation with 9-(4-formylbenzoylamino)-atc 6 and thiol-
Michael addition with the maleimido functionalized atc-derivative
7, respectively, proceeded easily in buffered solution and we ob-
tained the respective peptide-atc conjugates 10 and 11, in good
yields. In turn, both peptides were obtained as two isomers with
the main product eluting at the higher retention time. Interest-
ingly, the major isomer of the thiol-maleimide conjugate displayed
two sets of 1H NMR signals. We assume that the new stereogenic
center of the succinimide ring did not form in a stereoselective
manner and, thus, two diastereomers were generated.
To investigate the in vivo activity of the peptide–anhydrotetra-
cycline conjugates, HeLa cells were cotransfected with the reporter
plasmid pUHC13-38a expressing firefly luciferase under Tet control
and a plasmid expressing the transregulator rtTA-S3.7b This reverse
transactivator shows relaxed effector specificity, that is, it binds
tetracycline derivatives that are not or less efficiently recognized
by other Tet transregulators (Pook, E.; Krueger, C.; Berens, C.; Hil-
len, W. unpublished observations). For induction of reporter gene
expression, transfected cells were incubated 24 h with the respec-
Figure 2. (a) Schematic representation of the revTetR-transactivator fusion protein
rtTA binding to DNA in the presence of a tetracycline derivative; (b) revTetR binds
to DNA in the presence of a tc-peptide conjugate and transcription is triggered by
the transactivating peptide covalently bound to atc.
the attachment of suitable linkers for orthogonal ligation of unpro-
tected peptides via their N-termini. To determine cell penetration,
TetR interaction and transactivating potential of the conjugates, a
test system consisting of a Tet-dependent luciferase reporter gene
and the reverse transactivator rtTA-S37b was used. Moreover, ini-
tial structure–activity relationships should help establish an effec-
tive ligand-based regulatory system.
2. Results and discussion
To take advantage of the high binding and inducing activity of
anhydrotetracycline,13 our initial investigations were directed to-
wards a solid phase supported peptide synthesis and a subsequent
ligation in solution with a suitable atc-derivative to construct the
anhydrotetracycline–peptide conjugates. We were aware of puta-
tive synthetic problems which conjugation of a peptide with the
multifunctional tetracycline system might raise. To circumvent
side reactions, we pursued the direct acylation strategy using pro-
tected peptides (Scheme 1). Starting from chlorotritylchloride re-
sin, N-Fmoc-11-amino-3,6,9-trioxaundecanoic acid was attached
by alkylation. Subsequent deprotection of the linker gave the
loaded resin 1. Repetitive cycles of peptide coupling reactions were
performed using PyBOP/Fmoc-protected amino acids followed by
Fmoc-deprotection, with microwave irradiation promoting both
acylation and deprotection in a fast and effective way. Aspartic acid
side chains were incorporated as tert-butyl esters and terminal
amino acids were introduced as N-Boc protected derivatives. In
particular, Boc-Asp(OtBu)-OH was employed for the synthesis of
the VP1 sequence and Boc-NH(CH2CH2O)3CH2CO2H for VP2, which
should serve as solubility enhancer for the hexadecamer. The pep-
tide resins were cleaved with hexafluoroisopropanol to obtain the
fully protected peptide fragments 2 and 3 sufficiently pure for the
tive atc-derivative (5 lM). The data in Table 1 clearly show that the
atc-peptide conjugates investigated penetrate the cell membrane.
Furthermore, they bind to the reverse transactivator and trigger
its conformational change leading to DNA-binding and activation
of transcription (Fig. 2b). Structure–activity relationships can also
be deduced. Comparison of compounds 4, 10 and 11 demonstrates,
that the transactivating properties also strongly depended on the
nature of the linking element and the point of peptide attachment,
but not as significantly from the length of the peptide sequence.

S. Lochner et al. / Bioorg. Med. Chem. 18 (2010) 6127–6133
6129
O
(
O
)
HN
Fmoc
OH
3
O
a
b
(
O
)
ClTrt
Cl
H2N
O
ClTrt
3
1
O
O
O
O
(
O
)
BocNH-D(O)FD(O)LD(O)MLG
N
H
OH
OH
3
c,b,d
2
1
O
Boc
(
O
)
(
O
N
)
N
N-D(O)FD(O)LD(O)MLGD(O)FD(O)LD(O)MLG
H
N
H
3
3
H
3
CH3
OH
NH2
atc
e
O
O
O
H2N
(
(
O
)
N
H
N
H
2
3
H2N-DFDLDMLG
3
OH
O
O
f
O
OH OH
CH3
4 (18%)
N
atc
OH
O
O
H2N
e
f
(
O
NH2
)
O
)
H2N
N-DFDLDMLGDFDLDMLG
H
N
H
N
H
3
3
OH
O
O
O
OH OH
5 (6%)
Scheme 1. Reagents and conditions: (a) chlorotritylchloride resin, DIPEA, CH2Cl2, rt, 2 h, then successive washings with CH2Cl2/MeOH/DIPEA (17:2:1), CH2Cl2, DMF, CH2Cl2;
(b) piperidine/DMF (1:4), 18 cycles microwave irradiation 100 W, each ꢀ10 °C to 35 °C for 5 s; (c) Fmoc-AA-OH/PyBOP/HOBt/DIPEA (5:5:7.5:5), DMF, 15 cycles microwave
irradiation 50 W, each ꢀ10 °C to 35 °C for 10 s, N-terminal amino acid: Boc-Asp(OtBu)-OH (for peptide 2) or Boc-NH(CH2CH2O)3CH2CO2H (for peptide 3); (d)
hexafluoroisopropanol/CH2Cl2 (1:4), rt, 30 min; (e) (1) PyBOP, HOBt, DIPEA, NMP, rt, then 9-H2N-atc, 15 cycles microwave irradiation 100 W, each ꢀ10 °C to 35 °C for 10 s; (f)
TFA/CH2Cl2 (1:1), 0 °C to rt, 2–4.5 h, then RP 18-HPLC.
main. Addition of atc led only to a threefold increase in luciferase
activityover the basal level indicating that the mutations introduced
had indeed largely diminished VP16 domain functionality. Addition
of compound 5 resulted in a larger, ninefold increase in luciferase
activity demonstrating functionality of the peptide sequence in
the atc-peptide conjugate in activating transcription.
O
O
atc
atc
OH
OH
H2N
N
H
O
O
a
C
H
C
H
6 (55%)
atc
O
O
O
O
3. Conclusion
atc
H2N
N
N
N
H
b
In conclusion, bioconjugates of anhydrotetracycline and the
Herpes simplex derived minimal active sequences VP1 and VP2
were synthesized. Different ligation strategies were applied and a
biological evaluation in HeLa cells expressing the reverse transac-
tivator rtTA-S3 was performed. The data clearly displayed that (i)
the structure of the linker unit contributes strongly to the biologi-
cal activity of a conjugate, (ii) the atc-peptide conjugates are able
to penetrate the cell membrane, (iii) they are biologically active
as effectors for rtTA-S3 and that (iv) the VP2 sequence can activate
transcription when coupled to atc. This demonstrates the general
feasibility of ligand-based transactivation.
O
O
7 (56%)
Scheme 2. Reagents and conditions: (a) (1) SOCl2, DMFcat, toluene, CHCl3, 60 °C,
30 min; (2) NEt3, CH2Cl2, rt, 5 h; (b) HATU, DIPEA, NMP, rt, 3.5 h.
Thus, the N-terminally linked oxime derivative 10 proved excellent
activity when a 400-fold increase of luciferase activity indicated a
transcriptional activation substantially exceeding the inducing
properties of anhydrotetracycline. On the other hand, the thiol-
maleimide ligate 11 was almost devoid of function.
The 1.5 to 4-fold increased luciferase activity observed with com-
pounds 4, 5, and 10 with respect to atc suggested that the coupled
activating peptides contribute to overall transactivation. To address
this question more stringently, we inactivated the VP16 transactiva-
tion domain present in rtTA-S3 by mutagenesis.10a HeLa cells were
again cotransfected with the Tet-controlled reporter plasmid
4. Experimental
4.1. General
Reagents and solvents were obtained from Acros, Fluka, Aldrich
and Novabiochem, and were used as received. 2-Chlorotrityl resin
was purchased from IRIS Biotech or from Fluka. Fmoc-11-amino-
3,6,9-trioxaundecanoic acid (Fmoc-mini-PEG-3Ò) and Boc-11-ami-
pUHC13-38a and a plasmid expressing the variant rtTA-S3 with an
*
inactivated activation domain. Induction was determined after a
24 h incubation with either atc or compound 5 bearing the VP2 do-

6130
S. Lochner et al. / Bioorg. Med. Chem. 18 (2010) 6127–6133
O
a, b
O
(
)
NH
N
3NH2
H
O
Fmoc
Nle
Nle
O
O
N
H
O
N
H
(
)
HO
3 NHFmoc
O
O
O
atc
O
O
O
O
O
O
O
O
O
O
6, e
7, f
N
O
O
O
)
)
(
(
(
(
H
NH2
N
O
O
N
N
H
H2N
H2N
3 N
GLMDLDFD
H2N
H2N
3 N
GLMDLDFD
H
H
H
C
H
8
10 (52%)
a,c,d
O
O
O
atc
O
N
O
)
)
N
N
H
3 N
GLMDLDFD
N
GLMDLDFD
3 N
SH
H
H
S
H
H
O
9
11 (57%)
Scheme 3. Reagents and conditions: (a) piperidine/DMF (1:4), 18 cycles microwave irradiation 100 W, each ꢀ10 °C to 35 °C for 5 s; (b) (1) HATU, DIPEA, DMF, rt, 2 h, then 15
cycles microwave irradiation 50 W, each ꢀ10 °C to 35 °C for 10 s; (2) Ac2O, 2,6-lutidine, DMAP, rt, 15 min; (3) piperidine/DMF (1:4), 18 cycles microwave irradiation 100 W,
each ꢀ10 °C to 35 °C for 5 s; (c) Fmoc-AA-OH/PyBOP/HOBt/DIPEA (5:5:7.5:5), DMF, 15 cycles microwave irradiation 50 W, each ꢀ10 °C to 35 °C for 10 s, N-terminal amino
acid: Boc2NOCH2CO2H (for peptide 8) or Trt-SCH2CH2CO2H (for peptide 9); (d) phenol/H2O/thioanisole/triisopropysilane/TFA (0.25:0.25:0.25:0.125:0.15:5), rt, 2 h; (e) NaOAc/
HOAc buffer 0.1 M (pH 4.5), THF, CH3CN, rt, 20 h, then RP 18-HPLC; (f) KH2PO4/Na2HPO4 buffer (pH 5.5, Ph. Eur.), THF, CH3CN, rt, 200 min, then RP 18-HPLC.
Table 1
In vivo activity of peptide–anhydrotetracycline conjugates
Compound
Activitya
100
N
CH3
OH
NH2
atc
OH
O
O
O
O
OH OH
O
atc
(
O
)
4
5
150
190
N
N
H2N-DFDLDMLG
3
H
H
O
O
O
atc
O
(
O
)
(
N
O
)
H2N
H2N
N-(DFDLDMLG)2
H
N
3
3
H
H
atc
atc
O
O
O
N
H
10a
11
)
400
9
O
(
N
O
N
H
3 N
GLMDLDFD
H
C
H
O
O
O
O
O
N
O
(
)
N
H
N
H2N
3N
GLMDLDFD
S
H
H
O
Luciferase activities determined from cell extracts represent the means of triplicate samples with standard deviations given in relative light units (RLU; defined as relative
light units per g of total cell protein corrected for transfection efficiency).
% Activity with atc defined as 100%. Induction factors were calculated as RLUinducer/RLUbasal and varied in individual transfections between 107 and 525 for atc.
l
a
no-3,6,9-trioxaundecanoic acid ꢁ DCHA (Boc-mini-PEG-3Ò) were
purchased from Peptides International (Louisville, Kentucky) and
the DCHA salt was converted to the free acid according to the pro-
tocol of Peptides International. 3-Tritylsulfanyl-propionic acid was
obtained from Bachem (Bubendorf, Switzerland). Unless otherwise
noted, reactions were conducted without inert atmosphere. Micro-
wave assisted (DiscoverÒ microwave oven, CEM Corp.) peptide
synthesis was carried out in glass tubes loosely sealed with a sili-
con septum. Remark: the development of overpressure was
avoided by using DMF as the solvent and intermittent cooling.
Evaporations of final product solutions were done in vacuo with
a rotary evaporator or by lyophilization. Reactions were monitored
by HPLC-MS. ESI and APCI-MS were performed using a Bruker Es-
quire 2000 coupled with an Agilent 1100 analytic HPLC system.
The NMR spectra were recorded on a Bruker Avance 600 (1H at
600 MHz, 13C at 150 MHz) or
a
Bruker Avance 360 (1H at
360 MHz, 13C at 90 MHz) spectrometer. Chemical shifts are re-
ported relative to tetramethylsilane. Preparative RP-HPLC was per-
formed using Agilent 1100 preparative series, column: Zorbax
Eclipse XDB-C8, 21.2 ꢁ 150 mm, 5
lm particles [C8], flow rate
[FR] as specified below, eluent: methods P1–P3: 0.1% TFA in aceto-
nitrile (A) and 0.1% TFA in H2O (B) applying a gradient system as
described subsequently: P1: 25–42% A in 0–20 min, [FR] = 15 mL/
min. P2: 25–45% A in 0–20 min and then 45–100% A in 20–
23 min, [FR] = 16 mL/min. P3: 10–20% A in 0–5 min, 20–70% A in
5–20 min and then 70–95% A in 20–22 min, [FR] = 20 mL/min.

S. Lochner et al. / Bioorg. Med. Chem. 18 (2010) 6127–6133
6131
Purity and identity were assessed by analytical RP-HPLC (Agi-
lent 1100 analytical series, column: Zorbax Eclipse XDB-C8 analyt-
4.3. General procedure for the acylation of 9-aminoanhydrotet-
racycline with protected peptides and subsequent deprotection
ical column, 4.6 ꢁ 150 mm, 5
lm, flow rate: 0.5 ml/min, detection
wavelength: 254 nm) coupled to a Bruker Esquire 2000 mass
detector equipped with an ESI- or APCI-trap. Solvent system: x%
CH3OH in H2O (A) + 0.1% HCO2H (B): A1: 25–100% A in 0–12 min,
100–100% A in 12–18 min, 100–25% in 18–22 min. IR spectra were
measured on a Jasco 410 apparatus, using KBr.
The protected peptide, PyBOP and HOBtꢂH2O were dissolved in
dry NMP and diisopropylethylamine was added. After stirring for
10–15 min, 9-aminoanhydrotetracycline in dry NMP was added
and microwave irradiation (15 ꢁ 10 s, 100 W) with intermittent
cooling in an ethanol-ice bath was performed. The solvent was re-
moved and the protected peptide conjugate was either purified by
preparative RP-HPLC or isolated by precipitation with H2O. After
lyophilization of the HPLC fractions or drying of the precipitate,
the residue was dissolved in a chilled (0 °C) mixture of TFA/dichlo-
romethane 1:1 and then stirred at rt for 3–5 h. The solvent was re-
moved in vacuo and the residue was purified by RP-HPLC and
lyophilized.
4.2. Synthesis of fully protected peptides
4.2.1. 11-Amino-3,6,9-trioxaundecanoyl-2-Cl-Trt-resin (1)
2-Chlorotritylchloride resin (chloro-2-Cl-Trt-resin, loading:
1.1–1.6 mmol/g) was stirred in a solution of Fmoc-11-amino-
3,6,9-trioxaundecanoic acid (1.2 equiv)/diisopropylethylamine (DI-
PEA, 4 equiv) in CH2Cl2. After 2 h the resin was washed 3 ꢁ with
CH2Cl2/CH3OH/DIPEA (17:2:1), 3 ꢁ CH2Cl2, 2 ꢁ DMF, 2 ꢁ CH2Cl2
and dried in vacuo. The Fmoc group was removed by treating the
resin 1 ꢁ with 20% piperidine in DMF (microwave irradiation:
5 ꢁ 5 s, 100 W). In between each irradiation step, cooling of the
reaction mixture to a temperature of ꢀ10 °C was achieved by suf-
ficient agitation in an ethanol-ice bath. Finally, the resin was
washed with DMF (5ꢁ).
4.3.1. Conjugate 4ꢂ2CF3COOH
The compound was synthesized using the fully protected pep-
tide derivative Boc-NHD(OtBu)FD(OtBu)LD(OtBu)MLGCONH(CH2-
CH2O)3CH2CO2H (2, 26.7 mg, 0.019 mmol), PyBOP (10.2 mg,
0.019 mmol), HOBtꢂH2O (4.6 mg, 0.030 mmol), diisopropylethyl-
amine (201 ll, 0.119 mmol) and NMP (0.5 mL). Preactivation time:
10 min. Addition of 9-aminoanhydrotetracycline11 (12.9 mg, 0.029
mmol) in NMP (1.0 mL). Preparative RP-HPLC of the protected inter-
mediate (P1, tR: 14.3 min). Deprotection: TFA/CH2Cl2 1:1 (2 ml), 3 h.
Preparative RP-HPLC (P2) furnished a main product (tR: 12.9 min,
6.0 mg, 18%) and a second isomer (tR: 12.2 min, 6.0 mg, 7%), both
as an orange yellow powder. Analytical HPLC (A1): main product:
tr: 11.8 min, purity: 90% (contamination: 2nd isomer), second iso-
mer: tr: 11.0 min. ESI-MS: calcd for C70H96N12O25S [M+H]+: 1537.6,
[(M+2H)/2]: 769.3; found: 1537.7 [M+H]+, 769.6 [(M+2H)/2]+.
4.2.2. General procedure for the synthesis of protected peptides
a-Amino acids were incorporated as their commercially avail-
able derivatives: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Met-OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Phe-OH and, depending on the se-
quence, either Boc-Asp(OtBu)-OH or Boc-11-amino-3,6,9-trioxaun-
decanoic acid at the N-terminus. Peptide coupling was done
employing 5 equiv of the respective amino acid (or linker)/Py-
BOP/DIPEA and 7.5 equiv HOBt, dissolved in a minimum amount
of DMF (irradiation: 15 ꢁ 10 s, 50 W). In between each irradiation
step, cooling of the reaction mixture to a temperature of ꢀ10 °C
was achieved by sufficient agitation in an ethanol-ice bath. The
Fmoc group was removed as described above. After each coupling
and Fmoc-deprotection step, the resin was rinsed with DMF (4ꢁ).
After the last acylation step the N-terminal Fmoc-residue was
deprotected, the resin was rinsed with CH2Cl2 (10ꢁ) and dried in
vacuo. Cleavage from the resin was performed by the treatment
with hexafluoroisopropanol/dichloromethane 1:4 for 20 min fol-
lowed by filtration and removal of the solvent in vacuo. The follow-
ing crude peptides were obtained in sufficient purities and
employed for the acylation reactions without further purification.
4.3.2. Conjugate 5ꢂ2CF3COOH
The compound was synthesized using the fully protected pep-
tide derivative Boc-NH(CH2CH2O)3CONH[D(OtBu)FD(OtBu)LD(Ot-
Bu)MLG]2CONH(CH2CH2O)3CH2CO2H (3, 70.2 mg, 0.027 mmol),
PyBop (13.8 mg, 0.027 mmol), HOBtꢂH2O (6.2 mg, 0.041 mmol),
diisopropylethylamine (29 ll, 0.119 mmol) and NMP (1.0 mL). Pre-
activation time: 15 min (ultrasound bath). Addition of 9-aminoan-
hydrotetracycline11 (14.7 mg, 0.033 mmol) in NMP (1.0 mL). Then
the solvent was lyophilized, the residue redissolved in THF/meth-
anol 1:1 (6 mL) and the protected intermediate precipitated by
addition of H2O (10 mL), decanted and centrifugated. The mother
liquor was concentrated and the precipitation procedure was re-
peated with H2O (5 mL). The combined precipitates were washed
with H2O (5 mL), then again decanted, centrifugated and dried in
vacuo. Deprotection: TFA/dichloromethane 1:1 (4 ml), 5 h. Pre-
parative RP-HPLC (P2) furnished a main product (tR: 20.5 min,
4.2 mg, 6%) and a second isomer (tR: 19.9 min, 1.3 mg, 2%), both
as an orange yellow powder. Analytical HPLC (A1): main product:
tr: 13.1 min, purity: 88% (contamination: 2nd isomer), 2nd isomer:
tr: 12.5 min. 1H NMR (600 MHz, C5D5N, two sets of signals were ob-
served) d 0.63–0.71 (m, 3H, CH3 Leu), 0.72–1.16 (m, 21H, 7 ꢁ CH3
Leu), 1.79–1.86 (m, 1H, CbH Leu), 1.90–2.20 and 2.09, 2.11, 2.16,
4.2.3. Boc-Asp(OtBu)-Phe-Asp(OtBu)-Leu-Asp(OtBu)-Met-Leu-
Gly-11-amino-3,6,9-trioxa-undecanoic acid (2)
The fully protected peptide was synthesized according to the
general procedure starting from 11-amino-3,6,9-trioxaundeca-
noyl-2-Cl-Trt-resin (1). Boc-Asp(OtBu)-OH was introduced as the
last amino acid. For characterization, a small sample was fully
deprotected using TFA/dichloromethane 1:1. ESI-MS: calcd for
C
96H149N18O37S2 [M+H]+: 1114.5, found: 1114.5.
c
2.17 (m and 4 ꢁ s, 13H, 5 ꢁ CbH Leu, 2 ꢁ C H Leu and 2 ꢁ CH3
c
4.2.4. Boc-11-Amino-3,6,9-trioxaundecanoyl-Asp(OtBu)-Phe-Asp-
(OtBu)-Leu-Asp(OtBu)-Met-Leu-Gly-Asp(OtBu)-Phe-Asp(OtBu)-Leu-
Asp(OtBu)-Met-Leu-Gly-11-amino-3,6,9-trioxa-undecanoic acid (3)
The fully protected peptide was synthesized according to the
general procedure starting from 11-amino-3,6,9-trioxaundeca-
noyl-2-Cl-Trt-resin (1). Boc-11-amino-3,6,9-trioxaundecanoic acid
was introduced as the last amino acid. For characterization, a small
sample was fully deprotected using TFA/dichloromethane 1:1.
Met), 2.25–2.35 (m, 2H, 2 ꢁ C H Leu), 2.36–2.46 and 2.40 (m and
s, 5H, 2 ꢁ CbH Leu und ar-CH3), 2.47–2.61 and 2.58 (m and s, 7H,
CbH Met and N(CH3)2), 2.60–2.69 (m, 1H, CbH Met), 2.69–2.97
c
(m, 7H, 2 ꢁ CbH Met, 4 ꢁ C H Met and H-5), 3.06 (br d, 1H,
J = 10.2 Hz, H-4a), 3.20–3.96 and 3.70 (m, 41H, H-4, H-5, CH2 Gly,
5 ꢁ CH2CH2O, 13 ꢁ CbH Asp/Phe, linker OCH2CH2NHCO and OCH2-
CO), 4.11–4.41 and 4.33 (m, 7H, CH2 Gly, 3 ꢁ CbH Asp/Phe and
a
a
OCH2CO), 4.52–4.65 (m, 3H, 3 ꢁ C H AS), 4.74–4.81 (m, 1H, C H
C48H76N9O19S
[M+H]+: 2910.0, [(M+2H)/2]:
a
a
ESI-MS: calcd for
AS), 4.86–4.95 (m, 2H, 2 ꢁ C H AS), 4.95–5.29 (m, 10H, 8 ꢁ C H
AS and OCH2CH2NHCO), 7.25–7.28 (m, 1H, H-40 Phe within the pyr-
idine signal), 7.28–7.35 (m, 3H, H-40 Phe and H-30/50 Phe), 7.38–
1105.5, [(M+3H)/3]: 737.3; found: 1105.6 [(M+2H)/2]+, [(M+3H)/
3]: 737.6.

6132
S. Lochner et al. / Bioorg. Med. Chem. 18 (2010) 6127–6133
7.47 (m, 3H, H-7 and H-30/50 Phe), 7.48–7.54 (m, 2H, H-20/60 Phe),
7.60–7.69 (m, 3H, H-20/60 Phe and NH), 8.17–8.36 (m, 2H,
2 ꢁ NH), 8.54–8.71 (m, 3H, 3 ꢁ NH), 8.76–8.85 (m, 2H, NH and
H-8), 8.88–9.19 (m, 9H, 9 ꢁ NH), 9.59–9.70 und 9.64 (m and s,
2H, NH und NH-9), 10.11–10.22 (m, 2H, CONH2). ESI-MS: calcd
4.5. General procedure for the synthesis of N-terminally linked
peptides
The synthesis was accomplished starting from Rink-amide AM
resin (200–400 mesh, Novabiochem, loading: 0.25 mmol/g). As
the first amino acid, Fmoc-11-amino-3,6,9-trioxaundecanoic acid
(1 equiv) was attached employing 1 equiv PyBOP/DIPEA and
1.5 equiv HOBt, dissolved in a minimum amount of DMF, followed
by capping with a mixture of acetic acid anhydride/2,6-lutidine/
for
C
118H170N21O43S2 [M+H]+: 2633.1, [(M+2H)/2]: 1317.1,
[(M+3H)/3]: 878.4; found: 1317.2 [(M+2H)/2]+, [(M+3H)/3]:
879.0.
4.4. Synthesis of the 9-acylaminoanhydrotetracycline precursors
4-dimethylaminopyridine 5:6:1 in DMF. a-Amino acids were
incorporated as their commercially available derivatives: Fmoc-
Gly-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Phe-OH and, depending on the sequence, either Bis-Boc-ami-
no-oxyacetic acid or 3-tritylsulfanylpropionic acid were attached at
the N-terminus. Fmoc-deprotection and elongation was performed
as described above. Cleavage from the resin was performed by the
treatment with a mixture of TFA/phenol/H2O/thioanisole/EDT/
triisopropylsilane 80:5:5:5:3:2 for 2 h followed by filtration and re-
moval of the solvent in vacuo. The crude peptides were precipitated
in t-butylmethylether, centrifugated and washed with t-butylmeth-
ylether (4ꢁ). They were obtained in sufficient purities and employed
for the ligation reactions without further purification.
4.4.1. 9-(4-Formylbenzoylamino)-anhydrotetracyclineꢂCF3COOH (6)
To a solution of 4-formylbenzoic acid (35.8 mg, 0.238 mmol) in
a mixture of dry toluene (1.5 mL) and dry chloroform (1.5 mL) DMF
(15 lL) and thionyl chloride (80 lL, 1.096 mmol) were added drop-
wise at rt and then the mixture was stirred for 30 min at 60 °C. The
solvent was evaporated and the crude product was dried in vacuo.
9-Aminoanhydrotetracycline11 (105.1 mg, 0.238 mmol) was dis-
solved in dry dichloromethane (2 mL) and added to the crude
acid chloride, followed by the addition of triethylamine (33 ll,
0.237 mmol). After stirring for 5 h, the solvent was removed in
vacuo and the crude product purified by preparative RP-HPLC.
(P3, tR: 16.7 min) 1 to afford 90.5 mg (55%) as a red powder,
mp 173–176 °C (decomposition). Analytical HPLC (A1): tr:
15.8 min, purity: 97%. IR (KBr) 3600–2700, 1698, 1671, 1650,
4.5.1. Aminooxyacetyl-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly-11-
amino-3,6,9-trioxa-undecanoic amide (8)
1599 cmꢀ1
;
1H NMR (600 MHz, C5D5N): d 2.44 (s, 3H, ar-CH3),
The peptide was synthesized according to the general procedure.
2.62 (s, 6H, N(CH3)2), 3.12 (ddd, 1H, J = 11.7, 4.5, 1.9 Hz, H-4a),
3.59 (dd, 1H, J = 16.6, 4.5 Hz, H-5 ), 3.73–3.81 (m, 2H, H-4 and
ESI-MS: calcd for C50H80N11O20S [M+H]+: 1186.5, found: 1186.5.
a
H-5b), 7.54 (d, 1H, J = 9.1 Hz, H-7), 8.02–8.09 (m, 2H, H-30/50),
8.38–8.44 (m, 2H, H-20/60), 8.91 (d, 1H, J = 9.1 Hz, H-8), 10.11–
10.18 (br s and s, 2H, NH und CHO), 10.34 (br s, 1H, NH),
10.37 (br s, 1H, CONH2).13C NMR (150 MHz, C5D5N) d 14.6 (ar-
CH3), 25.4 (C-5), 42.5 (N(CH3)2), 42.6 (C-4a), 65.7 (C-4), 78.3 (C-
12a), 101.7 (C-2), 110.1 (C-11a), 113.5 (C-10a), 115.5 (C-7),
122.8 (C-6), 123.0 (C-9), 128.5 (C-8), 129.2 (C-20/60), 130.3 (C-
30/50), 132.0 (C-5a), 136.2 (C-6a), 137.1 (C-10), 139.2 (C-40),
141.3 (C-10), 165.8 (C-11), 166.6 (ar-NHCO), 175.0 (CONH2),
192.4 (ar-CHO), 194.1 (C-1), 198.2 (C-3), 200.0 (C-12). APCI-MS
574.7 [M+H]+.
4.5.2. 3-Sulfanylpropionyl-Asp-Phe-Asp-Leu-Asp-Met-Leu-Gly-
11-amino-3,6,9-trioxa-undecanoic amide (9)
The peptide was synthesized according to the general procedure.
ESI-MS: calcd for C51H81N10O19S2 [M+H]+: 1201.5, found: 1201.5.
4.6. Ligation
4.6.1. Conjugate 10ꢃCF3COOH
The peptide H2NOCH2CO–DFDLDMLG–NH(CH2CH2O)3CH2CO-
NH2 (8, 11.4 mg, 8.7 lmol) was dissolved in a mixture of CH3CN
(0.5 mL) and 0.1 M acetate buffer pH 4.5 (1.0 mL). To a suspension
of 9-(4-formylbenzoylamino)-anhydrotetracyclineꢂCF3COOH (6,
4.4.2. 9-(3-Maleimidopropionylamino)-anhydrotetracyclineꢂCF3-
6.0 mg, 8.7 lmol) in CH3CN (0.5 mL) and 0.1 M sodium acetate buf-
COOH (7)
fer pH 4.5 (1.0 mL) THF (q.s.) was added to result in the formation
of a clear solution. This solution was slowly added to the peptide
solution and stirred for 20 h under nitrogen. The solvent was re-
moved by lyophilization and preparative RP-HPLC (P3) was per-
formed to furnish a main product (tR: 15.9 min, 5.3 mg, 33%) and
a second isomer (tR: 15.6 min, 3.1 mg, 19%), both as an orange yel-
low powder. Analytical HPLC (A1): main product: tr: 13.1 min, pur-
ity: 95% (contamination: 2nd isomer), second isomer: tr: 12.5 min,
purity: 94%. 1H NMR (600 MHz, C5D5N) d 0.85 (d, 3H, J = 6.4 Hz, CH3
Leu), 0.88 (d, 6H, J = 6.0 Hz, CH3 Leu), 0.93 (d, 3H, J = 6.0 Hz, CH3
Leu), 1.92–1.98 (m, 2H, CbH2 Leu), 2.00–2.11 and 2.02 (m and s,
To
0.238 mmol) and HATU (62.7 mg, 0.165 mmol) in dry DMF
(0.5 mL) diisopropylethylamine (57 L, 0.332 mmol) was added
a
solution of 3-maleimidopropionic acid (27.8 mg,
l
dropwise at rt and then, after 15 min, a solution of 9-aminoanhy-
drotetracycline11 (72.9 mg, 0.165 mmol) in dry DMF (1.5 mL). After
stirring for 5 h, the solvent was removed in vacuo and the crude
product purified by preparative RP-HPLC (P3, tR: 15.0 min) 1 to af-
ford 65.4 mg (56%) as a red powder, mp 166–170 °C (decomposi-
tion). Analytical HPLC (A1): tr: 9.2 min, purity: 97%. IR (KBr)
3650–2900, 3103, 2954, 2922, 1772, 1710, 1674, 1640,
c
1599 cmꢀ1
;
1H NMR (600 MHz, C5D5N):d 2.40 (s, 3H, ar-CH3),
7H, CbH2 Leu, 2 ꢁ H Leu and SCH3), 2.44 (s, 3H, ar-CH3), 2.49 (m,
2.62 (s, 6H, N(CH3)2), 3.10 (ddd, 1H, J = 11.4, 4.5, 1.6 Hz, H-4a),
1H, Hb Met), 2.56–2.67 und 2.62 (m and s, 7H, Hb Met and
0
c
3.11 (t, 2H, J = 7.2 Hz, C2 H2), 3.55 (dd, 1H, J = 16.4, 4.5 Hz, H-
N(CH3)2), 2.82 (ddd, 1H, J = 13.3, 7.1, 6.1 Hz, H Met), 2.91 (ddd,
c
5
a
), 3.73 (dd, 1H, J = 16.4, 1.6 Hz, H-5b), 3.75 (d, 1H, J = 11.4 Hz,
1H, J = 13.3, 8.9, 5.0 Hz, H Met), 3.12 (ddd, 1H, J = 11.9, 4.7,
0
H-4), 4.18 (t, 2H, J = 7.2 Hz, C3 H2), 6.81 (s, 2H, maleinimide),
7.45 (d, 1H, J = 9.2 Hz, H-7), 8.83 (d, 1H, J = 9.2 Hz, H-8), 10.11–
2.1 Hz, H-4a), 3.19 (dd, 1H, J = 16.6, 6.6 Hz, Hb Asp1), 3.25 (dd,
1H, J = 16.5, 6.0 Hz, Hb Asp2), 3.30 (dd, 1H, J = 13.9, 8.7 Hz, Hb
Phe), 3.33 (dd, 1H, J = 16.3, 8.3 Hz, Hb Asp3), 3.37 (dd, 1H, J = 16.5,
7.0 Hz, Hb Asp2), 3.43 (dd, 1H, J = 16.3, 5.5 Hz, Hb Asp3), 3.44 (dd,
1H, J = 16.6, 7.4 Hz, Hb Asp1), 3.53 (dd, 1H, J = 13.9, 5.3 Hz, Hb
Phe), 3.56–3.64 und 3.59 (m and dd, 7H, J = 15.6, 4.7 Hz,
10.16 (m, 2H, NH-9 and CONH2), 10.37 (CONH2).13
C NMR
(150 MHz, C5D5N) d 14.6 (ar-CH3), 25.4 (C-5), 35.0 (C-20/30), 35.9
(C-20/30), 42.6 (N(CH3)2), 42.6 (C-4a), 65.8 (C-4), 78.3 (C-12a),
101.7 (C-2), 109.8 (C-11a), 113.2 (C-10a), 115.6 (C-7), 124.7 (C-
9), 128.7 (C-8), 131.4 (C-5a), 136.5 (C@C, imide), 136.7 (C-6a),
151.1 (C-10), 166.1 (C-11), 166.2 (C@O, imide), 170.0 (ar-NHCO),
175.1 (CONH2), 194.0 (C-1), 198.3 (C-3), 200.3 (C-12). APCI-MS
593.3 [M+H]+.
(OCH2CH2)3 and H-5a), 3.65–3.70 (m, 6H, (OCH2CH2)3), 3.76 (d,
1H, J = 11.9 Hz, H-4), 3.76 (dd, 1H, J = 15.6, 2.1 Hz, H-5b), 4.23 (s,
a
2H, H2NCOCH2O), 4.33 (dd, 1H, J = 16.5, 6.1 Hz, C H Gly), 4.36
a
a
(dd, 1H, J = 16.5, 5.9 Hz, C H Gly), 4.79 (m, 1H, C H Leu or Met),

S. Lochner et al. / Bioorg. Med. Chem. 18 (2010) 6127–6133
6133
a
4.89–5.00 and 4.91, 4.97 (m and 2 ꢁ d, 3H, 2 ꢁ J = 15.4 Hz, C H Leu
ified Eagle’s medium supplemented with 10% foetal bovine serum
(Gibco-BRL), 120 g/mL penicillin, 120 g/mL streptomycin and
2 mM -glutamine in a humidified incubator at 37 °C under 7.5%
a
or Met and C@N–OCH2CO), 5.06 (ddd, 1H, J = 9.6, 7.0, 4.5 Hz, C H
l
l
Leu or Met), 5.21 (ddd, 1H, J = 8.3, 7.4, 5.5 Hz, C H Asp3), 5.38
L
a
(ddd, 1H, J = 8.0, 7.0, 6.0 Hz, C H Asp2), 5.43 (m, 1H, C H Phe),
CO2. Transient transfections and luciferase activity determinations
a
a
5.55 (m, 1H, C H Asp1), 7.20 (m, 1H, H-40 Phe), 7.30 (m, 2H, H-20/
were performed as described.4b
a
60 Phe), 7.38 (m, 2H, H-30/50 Phe), 7.54 (d, 1H, J = 9.1 Hz, H-7),
7.73 (m, 2H, BB0), 7.87 (br s, 1H, CH2CONH2), 8.19 (s, 1H, N@CHPh),
8.24 (br s, 1H, CH2CONH2), 8.26 (m, 2H, AA0), 8.32–8.35 (m, 1H,
OCH2CH2NH), 8.57 (d, 1H, J = 7.2 Hz, NH), 8.71 (d, 1H, J = 7.2 Hz,
NH), 8.83 (dd, 1H, J = 6.1, 5.9 Hz, NH Gly), 8.93 (d, 1H, J = 9.1 Hz,
H-8), 8.95 (d, 1H, J = 7.0 Hz, NH), 9.20 (d, 1H, J = 8.0 Hz, NH Asp2),
9.26 (d, 1H, J = 6.2 Hz, NH), 9.48 (d, 1H, J = 7.4 Hz, NH Asp3), 9.54
(d, 1H, J = 7.0 Hz, NH), 10.09 (s, 1H, C9NH), 10.16 (br s, 1H, CONH2
*
4.8. Construction of rtTA-S3
The amino acid exchanges SL439, FG442, SL444 and FA475 were
introduced into the VP16 domain by site-directed mutagenesis
using overlap extension PCR,16c four mutagenic oligonucleotides
and two flanking oligonucleotides to introduce the restriction sites
for cloning. The resulting DNA-fragment was incubated with the
restriction enzymes XcmI/BamHI and cloned into likewise-re-
stricted pWHE330.4b The correct insertion was verified by sequenc-
ing. All plasmid and primer sequences are available upon request.
atc), 10.37 (br s, 1H, CONH2 atc). ESI-MS: calcd for C80H105N14O28
S
[M+H]+: 1741.7, [(M+2H)/2]: 871.4; found: 1741.7 [M+H]+, 871.7
[(M+2H)/2]+.
4.6.2. Conjugate 11ꢂCF3COOH
Acknowledgments
9-(3-Maleimidopropionylamino)-anhydrotetra-cyclineꢂCF3COOH
(7, 10.5 mg, 0.015 mmol) was dissolved in THF (volume least pos-
sible), then phosphate buffer pH 5.5, (Ph. Eur., 3.0 mL) was added.
The peptide HSCH2CH2O–DFDLDMLG–NH(CH2CH2O)3CH2CONH2
This work was supported by the Deutsche Forschungsgemeins-
chaft (SFB 473). Andreas Schmidt, Iris Torres and Michaela Kettler
are acknowledged for skillful technical assistance.
(8, 11.4 mg, 8.7 lmol) was dissolved in a mixture of CH3CN
(1.5 mL) and phosphate buffer pH 5.5 (Ph. Eur., 1.0 mL) and slowly
added to the solution described above. After 3 h stirring under
nitrogen, the solvent was removed by lyophilization and prepara-
tive RP-HPLC (P3) was performed to furnish a main product (tR:
References and notes
1. (a) Gossen, M.; Bujard, H. Annu. Rev. Genet. 2002, 36, 153; (b) Berens, C.; Hillen,
W. Eur. J. Biochem. 2003, 270, 3109; (c) Bertram, R.; Hillen, W. Microb.
Biotechnol. 2008, 1, 2.
14.9 min, 13.3 mg, 47%) and
a
second isomer (tR: 14.7 min,
2. (a) Mayford, M.; Bach, M. E.; Huang, Y. Y.; Wang, L.; Hawkins, R. D.; Kandel, E. R.
Science 1996, 274, 1678; (b) Sarkisian, C. J.; Keister, B. A.; Stairs, D. B.; Boxer, R.
B.; Moody, S. E.; Chodosh, L. A. Nat. Cell Biol. 2007, 9, 493; (c) Wernig, M.;
Lengner, C. J.; Hanna, J.; Lodato, M. A.; Steine, E.; Foreman, R.; Staerk, J.;
Markoulaki, S.; Jaenisch, R. Nat. Biotechnol. 2008, 26, 916.
2.7 mg, 10%), both as an orange yellow powder. Analytical HPLC
(A1): main product: tr: 13.7 min, purity: 91% (contamination: 6%
2nd isomer), second isomer: tr: 12.7 min, purity: 81% (contamina-
tion: 17% 1st isomer). 1H NMR (600 MHz, C5D5N, two sets of signals
were observed, ratio 1:1) d 0.86–0.92 (m, 9H, CH3 Leu), 0.92–0.97 (m,
3H, CH3 Leu), 1.91–2.15 and 2 ꢁ 2.03 (m and 2 ꢁ s, 9H, 2 ꢁ CbH2 Leu,
3. For total synthesis, see: Charest, M. G.; Siegel, J. D.; Myers, A. G. J. Am. Chem. Soc.
2005, 127, 8292.
4. (a) Krueger, C.; Pfleiderer, K.; Hillen, W.; Berens, C. BioTechniques 2004, 37, 546;
(b) Berens, C.; Lochner, S.; Löber, S.; Usai, I.; Schmidt, A.; Drueppel, L.; Hillen,
W.; Gmeiner, P. ChemBioChem 2006, 7, 1320; (c) Kormann, C.; Pimenta, I.;
Löber, S.; Wimmer, C.; Lanig, H.; Clark, T.; Hillen, W.; Gmeiner, P. ChemBioChem
2009, 10, 2924.
5. (a) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers, A. G. Science
2005, 308, 395. Charest, M. G.; Lerner, C. D.; (b) Brubaker, J. D.; Myers, A. G. Org.
Lett. 2007, 9, 3523.
c
2 ꢁ C H Leu and CH3 Met), 2.39 (s, 1.5H, ar-CH3), 2.40 (s, 1.5H, ar-
CH3), 2.47–2.55 (m, 1H, CbH Met), 2.58–2.64 and 2 ꢁ 2.61 (m and
2 ꢁ s, 7H, CbH Met and N(CH3)2), 2.76–2.89 (m, 4H, H-400, C H Met
c
c
and SCH2CH2CO), 2.92 (ddd, 0.5H, J = 13.3, 8.7, 4.8 Hz, C H Met),
c
2.93 (ddd, 0.5H, J = 13.5, 8.5. 5.1 Hz, C H Met), 3.05–3.17 (m, 4H,
6. (a) Shin, M. K.; Levorse, J. M.; Ingram, R. S.; Tilghman, S. M. Nature 1999, 402,
496; (b) Sigler, A.; Schubert, P.; Hillen, W.; Niederweis, M. Eur. J. Biochem. 2000,
267, 527.
a
H-4a, C H2 propionic acid and CbH Asp2), 3.20–3.48 (m, 9H, H-5
a,
SCH2CH2CO, 2 ꢁ H-4 succinimide ring, 2 ꢁ CbH Asp und CbH Phe),
a
and CbH Phe), 3.59–3.65 (m, 6H,
7. (a) Gossen, M.; Freundlieb, S.; Bender, G.; Müller, G.; Hillen, W.; Bujard, H.
Science 1995, 268, 1766; (b) Urlinger, S.; Baron, U.; Thellmann, M.; Hasan, M. T.;
Bujard, H.; Hillen, W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7963; (c) Zhou, X.;
Vink, M.; Klaver, B.; Berkhout, B.; Das, A. T. Gene Ther. 2006, 13, 1382.
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S.; Helbl, V.; Guthmann, J.; Pook, E.; Grimm, S.; Hillen, W. Gene 2000, 247, 103; (c)
Akagi, K.; Kanai, M.; Saya, H.; Kozu, T.; Berns, A. Nucleic Acids Res. 2001, 29, e23.
9. (a) Grbavec, D.; Stifani, S. Biochem. Biophys. Res. Commun. 1996, 223, 701; (b)
Molloy, D. P.; Milner, A. E.; Yakub, I. K.; Chinnadurai, G.; Gallimore, P. H.; Grand,
R. J. J. Biol. Chem. 1998, 273, 20867; (c) Frangioni, J. V.; LaRiccia, L. M.; Cantley, L.
C.; Montminy, M. R. Nat. Biotechnol. 2000, 18, 1080; (d) Lu, Z.; Ansari, A. Z.; Lu,
X.; Ogirala, A.; Ptashne, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 8591.
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883; (b) Tanaka, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4311; (c) Baron, U.;
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A. K.; Nguyen, D. H.; Dervan, P. D.; Ptashne, M. Chem. Biol. 2001, 8, 583.
11. (a) Orth, P.; Schnappinger, D.; Sum, P. E.; Ellestad, G. A.; Hillen, W.; Saenger,
W.; Hinrichs, W. J. Mol. Biol. 1999, 285, 455; (b) Sum, A. T.; Petersen, P. J. Bioorg.
Med. Chem. Lett. 1999, 9, 5071. and references cited therein; (c) Sum, P. E.; Ross,
A. T.; Petersen, P. J.; Testa, R. T. Bioorg. Med. Chem. Lett. 2006, 16, 1449. and
references cited therein; (d) Chen, C.-p.; Zeiger, A. R.; Wickstrom, E. Bioorg.
Med. Chem. Lett. 2007, 17, 6558.
3.51–3.58 (m, 2H, H-5
(OCH2CH2)3), 3.64–3.77 (m, 8H, (OCH2CH2)3, H-4 and H-5b), 4.15–
4.25 (m, 5H, CH2CONH2, CbH2 propionic acid and H-3 succinimide
ring), 4.30–4.39 (m, 2H, CH2 Gly), 4.74–4.81 (m, 1H, C H Leu1),
a
a
a
4.93–5.00 (m, 1H, C H Leu2), 5.02–5.09 (m, 1H, C H Met), 5.14–
a
a
5.21 (m, 1H, C H Phe), 5.33–5.40 (m, 1H, C H Asp3), 5.40–5.45 (m,
a
a
1H, C H Asp2), 5.45–5.52 (m, 1H, C H Asp1), 7.19–7.23 (m, 1H, H-
40 Phe), 7.27–7.32 (m, 2H, H-20/60 Phe), 7.37–7.43 (m, 2H, H-30/50
Phe), 7.45 (d, 1H, J = 9.1 Hz, H-7), 7.87 (br s, 1H, CH2CONH2), 8.24
(br s, 1H, CH2CONH2), 8.28–8.32 (m, 1H, OCH2CH2NH), 8.51–8.57
(m, 1H, NH Leu2), 8.66–8.71 (m, 1H, NH Met), 8.78–8.85 and 8.82
(m and d, 2H, J = 9.1 Hz, NH Gly and H-8), 8.92 (d, 0.5H, J = 6.0 Hz,
NH Asp3), 8.92 (d, 0.5 Hz, J = 6.4 Hz, Asp3), 9.03 (d, 0.5 Hz,
J = 6.4 Hz, NH Leu1), 9.07 (d, 0.5H, J = 6.0 Hz, NH Leu1), 9.26 (d,
0.5H, J = 7.2 Hz, NH Asp2), 9.30 (d, 0.5H, J = 7.2 Hz, NH Asp2), 9.55
(d, 0.5H, J = 6.8 Hz, NH Phe), 9.57 (d, 0.5H, J = 6.4 Hz, NH Phe), 9.57
(d, 0.5H, J = 7.6 Hz, NH Asp1), 9.60 (d, 0.5H, J = 7.6 Hz, NH Asp1),
10.06 (s, 0.5H, NH-9), 10.08 (s, 0.5H, NH-9), 10.14 (d, 1H, J = 1.6 Hz,
CONH2 Atc), 10.36 (d, 1H, J = 1.6 Hz, CONH2 Atc).
12. Menachery, M. D.; Cava, M. P. Can. J. Chem. 1987, 62, 2583.
13. (a) Degenkolb, J.; Takahashi, M.; Ellestad, G. A.; Hillen, W. Antimicrob. Agents
Chemother. 1991, 35, 1591; (b) Lederer, T.; Kintrup, M.; Takahashi, M.; Sum, P.
E.; Ellestad, G. A.; Hillen, W. Biochemistry 1996, 35, 7439.
14. For example, see: Spetzler, J. C.; Hoeg-Jensen, T. J. Peptide Sci. 2001, 7, 537.
15. For example, see: Liang, J. F.; Yang, V. C. Bioorg. Med. Chem. Lett. 2005, 15, 5071.
and references cited therein.
16. (a)Focus1986, 8, 9.; (b)Sambrook, J.;Russell, D. W. Molecular Cloning: ALaboratory
Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, USA,
2000; (c) Higuchi, R.; Krummel, B.; Saiki, R. Nucleic Acids Res. 1988, 16, 7351.
4.7. Bacterial strain, human cell line and cell culture methods
The bacterial strain DH5a16a was used for general cloning pro-
cedures16b and is derived from Escherichia coli K12. The cell line
HeLa (ATCC #CCL-2) was cultured in high glucose Dulbecco’s Mod-