Click-chemistry-derived tetracycline-amino acid conjugates exhibiting exceptional potency and exclusive recognition of the reverse tet repressor

Article abstract of DOI:10.1002/cbic.200900710

A click-chemistry-based synthesis of biologically active doxycycline-amino acid conjugates is described. Starting from 9-aminodoxycycline derivatives and complementary functionalized amino acids, ligation was accomplished by copper(I)-catalyzed azide-alky

Full text of DOI:10.1002/cbic.200900710

DOI: 10.1002/cbic.200900710
Click-Chemistry-Derived Tetracycline–Amino Acid
Conjugates Exhibiting Exceptional Potency and Exclusive
Recognition of the Reverse Tet Repressor
Igor Usai,[a] Marcus Krueger,[b] Jꢀrgen Einsiedel,[a] Wolfgang Hillen,[b] and Peter Gmeiner*[a]
A click-chemistry-based synthesis of biologically active doxycy-
cline–amino acid conjugates is described. Starting from 9-ami-
nodoxycycline derivatives and complementary functionalized
amino acids, ligation was accomplished by copper(I)-catalyzed
azide–alkyne [3+2] cycloaddition (CuAAC). The final products
were tested in a variety of TetR and revTetR systems, and the
C-terminally linked phenylalanine conjugate 12c exhibited
high selectivity for revTetR over TetR. Besides the unique prop-
erty of the specific effector 12c to effectively differentiate TetR
and its reverse phenotype, the test compound proved to be
almost devoid of any antibacterial activity; this will be highly
beneficial for future applications to control gene expression in
bacterial systems.
Introduction
Complementing genetic knockout strategies, receptors activat-
ed solely by synthetic ligands (RASSLs) and artificial regulatory
systems have been created as powerful tools in the new area
of synthetic biology. Thus, orthogonal ligand–protein pairs
have been engineered for nuclear hormone receptors,[1] and G
protein-coupled receptors activated solely by synthetic ligands
have been implemented as a new means to study neurotrans-
mission and signaling in vitro and in vivo.[2] As an example, a
GPCR–ligand pair that simulates the activation of D2L by dopa-
mine was engineered in our laboratory.[3] The Tet repressor pro-
tein (TetR) was originally discovered as a regulating instance in
many bacteria that efficiently blocks transcription by tightly
binding to the operator tetO. Exposure of a microorganism to
tetracycline effects dissociation of TetR, and therefore, expres-
sion of the transporter protein TetA, which mediates tetracy-
cline resistance. This principle proved to be advantageous to
control gene expression in pro- and eukaryotes. Applications
range from fermentation procedures to use in medical research
and gene therapy.[4] As a completely orthogonal regulatory
system, we constructed a mutant of the tetracycline-inducible
repressor protein TetR with specificity for the synthetic tetracy-
cline analogue 4-de(dimethylamino)anhydrotetracycline, which
was shown not to be effective at the wild-type protein.[5] In a
search for nonantibiotic tetracycline surrogates, the diarylpro-
pane-1,3-dione motif was identified as the minimal substruc-
ture responsible for the induction of TetR by tetracyclines.[6]
The binding of a TetR system displaying a reverse phenotype
(revTetR) to the DNA is effected in presence of tetracycline.[7]
Combining a chemical approach that diversifies the inducer
with a biological strategy that adjusts the regulatory protein to
a newly synthesized compound, the discovery of subtype-se-
lective tetracycline agonists capable of differentiating between
two reverse transactivators could be performed.[8] Aiming to
selectively activate revTetR in presence of wild-type TetR, we
planned to further manipulate the tetracycline structure. By
employing the recently described glycylcyclines[9] and TetR-in-
ducing peptides[10] as lead structures, we envisioned to extend
the pharmacophoric system of doxycycline by different amino
acids; this might favour revTet recognition or perturb binding
to specific sites of TetR and, thus, induce variant selectivity.
Because the crystal structure of tetR/glycylcycline complexes
showed a tunnel-like cavity around position 9 of the tetracy-
cline core,[9b] we decided to exploit 9-substituted tetracycline
derivatives to perform the bioconjugation. Taking advantage of
the concept of click chemistry,[11] we planned to further rely on
this versatile methodology for the conjugation of suitably
modified doxycycline derivatives. Starting from 9-aminodoxy-
cycline (H2N-dox), which has proven useful for the synthesis of
subtype selective tetracycline agonists,[12] acylation of the aro-
matic amino group with azido-substituted or alkynyl carboxylic
acids furnished precursors for the envisioned click-chemistry-
based ligation. Subsequently, suitably functionalized, amino-
acid-derived building blocks should be synthesized and sub-
jected to a Cu-promoted [3+2] cycloaddition (CuAAC) with a
complementary substituted H2N-dox derivative (Scheme 1). In
this publication, we present a microwave-assisted synthesis of
doxycycline–amino acid conjugates by click chemistry and the
biological evaluation in TetR and revTetR expressing systems.
[a] Dr. I. Usai, Dr. J. Einsiedel, Prof. Dr. P. Gmeiner
Department of Chemistry and Pharmacy, Emil Fischer Center
Chair of Medicinal Chemistry, Friedrich Alexander University
Schuhstrasse 19, 91052 Erlangen (Germany)
Fax: (+49)9131-8522585
E-mail: peter.gmeiner@medchem.uni-erlangen.de
[b] M. Krueger, Prof. Dr. W. Hillen
Department of Biology, Chair of Microbiology,
Friedrich Alexander University
Staudtstrasse 5, 91058 Erlangen (Germany)
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P. Gmeiner et al.
in DMF. Peptide coupling was performed with (benzotriazol-
oxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP)
and 1-hydroxybenzotriazole (HOBt) in presence of N-ethyldiiso-
propylamine (DIPEA). Cleavage from the resin was carried out
with 20% hexafluoroisopropanol in dichloromethane to furnish
the N-alkynoylamino acids 4a–c and azidovaleroyl derivatives
5a,b (Scheme 3).
Scheme 1. Concept of the doxycycline–amino acid ligation based on click
chemistry.
Results and Discussion
Scheme 3. Reagents and conditions: a) Fmoc-AA-OH, DIPEA, CH2Cl2, RT, 2 h;
b) piperidine/DMF 1:5, m-wave: 100 W, 5ꢂ5 s, 5ꢂꢀ10!308C; c) i. PyBOP,
HOBt, DIPEA, DMF, m-wave: 15ꢂ10 s, 50 W, 15ꢂꢀ10!308C; ii. hexafluoro-
isopropanol/CH2Cl2, RT, 2 h.
Synthesis
To obtain doxycycline derivatives presenting either an alkyne
or an azide functionality, we chose to start from 9-aminodoxy-
cyline, which should be acylated at the primary amine function
(Scheme 2).[13] Employing either a mixed or a symmetric anhy-
dride activation for the coupling of hexynoic acid or 5-azido-
pentanoic acid,[14] respectively, we were able to generate the
synthetic intermediates 1 and 2 in 23 and 92% yield, respec-
tively. Purification by preparative HPLC was necessary in order
to remove impurities resulting from 7-aminodoxycycline, which
was formed as a side product during the nitration–reduction
process of doxycycline.
When starting the first model reactions for the copper cata-
lyzed bioconjugation, we recognized that several problems
had to be addressed to facilitate the reaction of the doxycy-
cline precursors with the amino acid derivatives. At first, a suit-
able solvent system for all reactants and reagents had to be
established. Furthermore, we were conscious that formation of
a copper complex with both the tetracycline and the amino
acid derivative might impede the reaction by precipitation or
by sequestration of the catalyst. Additionally, tetracycline deriv-
atives are highly sensitive molecules in terms of light, oxida-
tion, high temperatures or extreme pH values. Therefore, we
had to find conditions to promote the cycloaddition reaction
without risking concomitant degradation.[15] In fact, screening
of a number of reaction conditions led us to a methodology
that combines the reactive components CuI (13 equiv), ascor-
bic acid (7 equiv) and DIPEA (17 equiv)[16,17] with microwave ir-
radiation;[18,19] this facilitated complete reaction of the N-acyl-
doxycycline derivatives 1 and 2 with the complementary func-
tionalized amino acid derivatives 5a,b and 4a–b, respectively,
within 5 min. After lyophilization of the solvent, the crude
products were dissolved in THF/HCl in order to cleave copper
complexes. The solution was finally subjected to preparative
HPLC to obtain the conjugates 6a,b and 8a–c in 74 to >99%
purities. Treatment of the conjugates 6a,b and 8b,c in conc.
methanol/HCl followed by HPLC gave the methyl esters 7a,b
and 9a,b, respectively (Scheme 4).
Nitrogen-linked amino acid derivatives were synthesized
starting from chlorotrityl-chloride resin. The resin was loaded
with Fmoc-protected alanine, phenylalanine, and glycine and
subsequently deprotected by treatment with 20% piperidine
Scheme 2. Reagents and conditions: a) i. iPr-COCl, NEt3, CH2Cl2, RT, 12 h; ii.
H2N-dox, NaHCO3, DMF, RT, 2 h. b) i. N3-(CH2)4-CO2H, DCC, CH2Cl2, 08C,
30 min, ii. ꢀ208C, 1 h; iii. H2N-dox, NaHCO3, DMF, RT, 2 h.
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Tetracycline–Amino Acid Conjugates
was performed and the C-terminally linked amino acid–doxycy-
cline conjugates 12a–d were obtained in 86 to >95% purities
(Scheme 6).
Scheme 4. Reagents and conditions: a) CuI (13 equiv), ascorbic acid
(7 equiv), DIPEA (17 equiv), DMF, m-wave: 100 W, 4ꢂ 30 s, 5ꢂꢀ10!508C;
b) MeOH/HCl conc. 10:1, RT, 14 h.
Scheme 6. Reagents and conditions: a) CuI (13 equiv), ascorbic acid
(7 equiv), DIPEA (17 equiv), DMF, m-wave: 100 W, 4ꢂ30 s, 5ꢂꢀ10!508C.
For the synthesis of the carboxy-linked doxycycline–amino
acid conjugates, we started from N,N-dimethylglycine (10a), N-
Boc-(S)-alanine (10b), N-Boc-(S)-phenylalanine (10c) and N-Boc-
N-(S)-alaninyl-glycine dipeptide (10b). Activation with N-(3-di-
methylaminopropyl)-N’-ethylcarbodiimide (EDC) in the pres-
ence of 1-hydroxy-7-azabenzotriazole (HOAt) and subsequent
aminolysis of the active ester intermediate with 3-amino-1-azi-
dopropane[20] furnished the coupled products 11a,b1,c1,d1.
Deprotection of 11 b1–11 d1 with trifluoroacetic acid led to for-
mation of the key intermediates 11 b2–11 d2, which could be
employed without further purification (Scheme 5).
The test compounds 6a,b, 7a,b, 8a–c and 12a–d were in-
vestigated for their ability to induce TetR or to corepress the
revTetR phenotype. For comparison, the biological data of tet-
racycline, doxycycline, and anhydrotetracycline at 0.4 mm are
given (Table 1). For the measurements, our test compounds
were employed at a concentration of 1 mm. Escherichia coli
WH207ltet50[21] and the “leaky” mutant NR698ltet50[6] were
the reporter strains used in this assay. The “leaky” strain E. coli
NR698 contains a mutation in the imp gene that allows in-
creased diffusion of low-molecular-weight effectors through
the outer membrane.[22] This leads to an increased intracellular
concentration of the tetracycline derivatives and prevents their
possible exclusion by the outer membrane observed in earlier
experiments. The activities of 13 test compounds were evaluat-
ed relative to 100% b-galactosidase activity, defined by a strain
lacking TetR. In this screening program, we were especially
looking for a high corepression of the revTetR system; during
this screen, the phenylalanine derivatives 6b, 9a and 12c par-
ticularly attracted our attention. Compound 6b, which is a
combination of hexynoyl-aminodoxycycline (1) and the N-ter-
minally linked azido acid 5b, displayed the most pronounced
revTetR mediated repression of the b-galactosidase activity for
the “leaky” E. coli mutant NR698ltet50 (3.5%ꢁ0.3%). Interest-
ingly, this conjugate proved equipotent in the “normal” strain
WH207ltet50 (4.3%ꢁ0.6%); this is obviously due to the ability
to cross the outer membrane. However, this biological effect is
not selective for revTetR, because significant dissociation of
TetR from tetO is triggered, as represented by the b-galactosi-
dase activity in the “normal” (37%ꢁ1%) and the “leaky”
(67%ꢁ4%) strains. Nearly identical results were obtained for
the TetR S135L[23] mutant with relaxed inducer specificity
(normal: 34%ꢁ2%, “leaky”: 69%ꢁ1%). Analogous behavior
could be observed for the phenylalanine ester derivative 9a,
which displays the inverse linker/triazole pattern. Observing
Starting from the doxycycline derivative 1, CuI-assisted [3+2]
cycloaddition with the amino acid derivatives 11a,b2,c2,d2
Scheme 5. Reagents and conditions: a) EDC, HOAt, NEt3, DMF, 08C, 30 min,
RT, 14 h; b) TFA/CH2Cl2 1:1, RT, 1 h.
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705

P. Gmeiner et al.
the revTetR(BD)-mediated corepres-
sion, strongly reduced b-galactosidase
activities of 6.5%ꢁ1.6% (“normal”
strain) or 4.5%ꢁ0.1% (“leaky”
mutant) were detected, while TetR in-
duction in both bacterial strains
showed parallel results (32%ꢁ3% vs.
56%ꢁ15% in “normal” vs. “leaky”, re-
spectively,
and
correspondingly,
33%ꢁ1% vs. 48%ꢁ7% for TetR
S135L). Notably, the TetR-i2 mutant
(TetR H64K S135L S138I), that exhibits
altered inducer specificity for 4-de(di-
methylamino)tetracyclines,[24] was not
significantly induced by both ligands
6b and 9a. Different behavior was
displayed by the C-terminally linked
Phe derivative 12c. For the
WH207ltet50, 12c was not active at
TetR (1.8%ꢁ0.1%) and only margin-
ally active at revTetR (58%ꢁ1%), but
in the “leaky” mutant, a very signifi-
cant reduction of the b-galactosidase
activity was detected (9.0%ꢁ0.2%).
Investigating TetR(BD) in the “leaky
mutant”, the induction remained at
the basal level (1.1%ꢁ0.0%). Closer
inspection of 12c in a reverse variant
of TetR(B)[25] revealed that the remain-
ing b-galactosidase expression was
even lower (2.6%ꢁ0.2%), while
TetR(B) (1.7%ꢁ0.2%) and the other
TetR mutants were not affected. Inter-
estingly, the biological activity of the
test compounds strongly depends on
the presence of a phenyl residue.
Thus, the alanine and glycine deriva-
tives 6a, 7a, 8a,b,c, 9b and 12a,b,d
displayed only poor to moderate po-
tency. For the phenylalanine conju-
gate 8b, we assume that penetration
of the bacterial lipid membrane is not
possible, as the ester derivative 9a is
active. In addition to their TetR induc-
tion capability, the toxicity of our tet-
racycline analogues was characterized
because an ideal small molecule in-
ducer of a TetR-regulated gene ex-
pression system should preferably
lack antibiotic effects. Obviously, the
outer cell wall serves as a primary
barrier of bacteria against antibiotic
substances. The test compounds
were investigated by minimal inhibi-
tory concentration (MIC) tests in the
two
different
E. coli
strains
WH207ltet50 (“normal”) or the “leaky”
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Tetracycline–Amino Acid Conjugates
by using Agilent 1100 preparative series, column: Zorbax Eclipse
XDB-C8, 21.2ꢂ150 mm, 5 mm particles [C8], flow rate (FR):
20 mLminꢀ1, eluent: solvent A: TFA (0.1%) in acetonitrile and sol-
vent B: TFA (0.1%) in H2O applying a gradient system as described
subsequently: P1: 10–10% A in 0–2 min and then 10–40% A in 2–
20 min. P2: 5–5% A in 1 min and then 5–64% A in 1–17 min.
mutant NR698ltet50, furthermore in Bacillus subtilis representa-
tive for gram-positive cells. The results are summarized in
Table 1. For E. coli WH207ltet50, the screening hits 6b, 9a and
12c exhibited a low antibacterial activity of 64 mgLꢀ1, while
the “leaky” mutant was more susceptible (32 mgLꢀ1 for all
compounds). In B. subtilis, the MIC was determined to be
64 mgLꢀ1 for 6b and 12c; interestingly, for 9a, the sensitivity
decreased to 16 mgLꢀ1. This might be due to a higher penetra-
tion of the ester derivative through the cell membrane.
Purity and identity were assessed by analytical RP-HPLC (Agilent
1100 analytical series, column: Zorbax Eclipse XDB-C8 analytical
column, 4.6ꢂ150 mm, 5 mm, FR: 0.5 mLminꢀ1, detection wave-
length: 254 nm) coupled to a Bruker Esquire 2000 mass detector
equipped with an ESI- or APCI-trap. Solvent system: solvent A:
CH3OH in H2O and solvent B: HCO2H (0.1%) in H2O applying a gra-
dient system as described subsequently: 10–10% A in 0–3 min,
10–100% A in 3–20 min, 100–100% A in 20–24 min. IR spectra
were measured on a Jasco 410 apparatus, by using a film on a
NaCl crystal.
Conclusions
Doxycycline–amino acid conjugates were synthesized. Ligation
was accomplished by [3+2] cycloaddition of azide and alkyne
substituted precursors, which were synthesized starting from
9-aminodoxycycline and functionalized amino acids. It was
possible to overcome the problems associated with tetracy-
cline or amino acid complexation by CuI. To discover their
effect on the tetracycline repressor, thirteen of the novel doxy-
cycline–amino acid conjugates were tested with TetR and re-
vTetR variants. The C-terminally linked amino acid conjugate
12c turned out to be the first tetracycline derivative that
exhibits high selectivity for revTetR over TetR. In addition, the
test compound proved to be almost devoid of any antibacteri-
al activity; which will be highly beneficial for applications to
control gene expression in bacterial systems. By exploiting the
capability to ligate unprotected peptide derivatives with tetra-
cycline derived effectors by click chemistry, we are pursuing
the synthesis of ligand-based trans-activating and trans-repres-
sing entities.
9-(5-Hexynoylamino)-doxycycline (1): 5-Hexynoic acid (0.072 mL,
0.65 mmol) and isobutyl acid chloride (0.068 mL, 0.65 mmol) were
dissolved in dry dichloromethane (4 mL). DIPEA (0.78 mmol,
0.128 mL) was added dropwise and the reaction stirred at RT over-
night. The solution was diluted with hexane (50 mL), washed 3ꢂ
with 0.5 n HCl (10 mL) dried with MgSO4, filtered and concentrated
in vacuo. The raw material was dissolved in DMF (dimethylforma-
mide, 2 mL) and slowly added to a solution of 9-aminodoxycy-
cline[13] (100 mg 0.22 mmol) and NaHCO3 (36.6 mg, 0.43 mmol) in
dry DMF (5 mL). After 1 h stirring at RT, the reaction was consid-
ered complete according to LC-MS analysis. The solvent was re-
moved in vacuo and the residue was purified by RP-HPLC (C8
using a mixture of solvent A: acetonitrile and solvent B: TFA (0.1%)
in H2O, 20–20% A in 0–10 min and then 20–75% A in 10–16 min,
FR: 20 mLminꢀ1, tR: 13.9 min) to afford 27.3 mg (23%) 1 as a
yellow powder. Analytical HPLC: tR: 15.8 min, purity: >99%;
1H NMR (600 MHz, CD3OD): d=1.54 (d, J=6.8 Hz, 3H), 1.91 (m,
2H), 2.26–2.33 (m, 3H), 2.57 (dd, J=12.0, 8.3 Hz, 1H), 2.59 (t, J=
7.6 Hz, 2H), 2.75 (m, 1H), 2.82 (d, J=11.3, 1H), 2.95 (s, 6H), 3.56
(dd, J=11.3, 8.3 Hz, 1H), 4.41 (s, 1H), 6.93 (d, J=8.3 Hz, 1H), 8.15
(d, J=8.3 Hz, 1H); 13C NMR (150 MHz, CD3OD): d=16.1, 18.6, 25.8,
36.4, 39.8, 43.0, 47.9, 48.0, 67.1, 70.0, 70.3, 74.7, 84.2, 96.2, 108.5,
116.1, 116.9, 126.6, 130.2, 144.5, 154.1, 172.9, 174.0, 174.1, 188.1,
195.6; IR (neat): n˜ =3530–3100, 2103, 1672, 1616 cmꢀ1; APCI-MS
(m/z): calcd for C28H32N3O9: 554.2 [M+H]+, found: 537.2
[M+HꢀOH]+.
Experimental Section
General: Reagents and solvents were obtained from Acros, Fluka,
Aldrich and Novabiochem, and were used as received. Doxycycline
was a generous gift from Excella Pharma (Feucht, Germany). 2-
Chlorotrityl resin was purchased from IRIS Biotech (Marktredwitz,
Germany). Unless otherwise noted, reactions were conducted with-
out inert atmosphere. Microwave-assisted (Discover microwave
oven, CEM Corp., (Kamp-Lintfort, Germany)) peptide synthesis was
carried out in glass tubes loosely sealed with a silicon septum.
Note: the development of overpressure was avoided by using DMF
as the solvent and intermittent cooling. Microwave promotion (Ini-
tiator microwave oven, Biotage Corp., (Uppsala, Sweden)) of the
cycloaddition reactions was carried out under nitrogen in com-
pletely sealed reaction vials. Evaporations of final product solutions
were done in vacuo with a rotary evaporator or by lyophilization.
Reactions were monitored by HPLC-MS or in case of peptides 11 a,
11 b1–d1 by TLC on Merck 60 F254 silica gel plates (0.25 mm), vi-
sualized by UV light, iodine and/or ninhydrin solution. Flash chro-
matography was carried out with 230–400 mesh silica gel and 0.8–
1.0 bar nitrogen pressure. Mass spectra were performed by EI ioni-
zation (70 eV) with solid inlet, EI-HRMS were obtained employing
peak matching M/DM=5000. ESI and APCI-MS was performed
using a Bruker Esquire 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 reported
relative to tetramethylsilane. Preparative RP-HPLC was performed
9-(5-Azidopentanoylamino)-doxycycline (2): Dicyclohexyl carbodi-
imide (103.2 mg, 0.5 mmol) in dichloromethane (1 mL) was added
dropwise at 08C to
a
solution of 5-azidopentanoic acid[14]
(143.1 mg, 1 mmol) in dichloromethane (5 mL) and the mixture
was stirred for 30 min. After cooling to ꢀ208C for 1 h, the precipi-
tate was removed by filtration and the solvent was evaporated.
The resulting symmetric anhydride was dissolved in DMF/dichloro-
methane (1:1, 2 mL) and added to a solution of 9-aminodoxycy-
cline[13] (200 mg, 0.435 mmol) and NaHCO3 (87.0 mg, 0.73 mmol) in
dry DMF (5 mL). After 2 h stirring at RT, the reaction was consid-
ered complete according to LC-MS analysis. The solvent was re-
moved in vacuo and the residue was purified by RP-HPLC (C8
using a mixture of solvent A: acetonitrile and solvent B: TFA (0.1%)
in H2O, 10–10% A in 2 min and then 10–80% A in 2–20 min, FR:
20 mLminꢀ1, tR: 13.3 min) to afford 27.3 mg (23%) 2 as a yellow
powder. Analytical HPLC: tR: 17.1 min, purity: >99%. 1H NMR
(600 MHz, CD3OD): d=1.54 (d, J=6.5 Hz, 3H), 1.69 (tt, J=7.4,
7.2 Hz, 2H), 1.79 (tt, J=7.5, 7.5 Hz, 2H), 2.51 (t, J=7.6 Hz, 2H), 2.56
(dd, J=12.3, 8.5 Hz, 1H), 2.74 (dq, J=13.2, 6.5 Hz, 1H), 2.81 (d, J=
11.4, 1H), 2.95 (s, 6H), 3.36 (t J=6.8 Hz, 2H), 3.56 (dd, J=11.4,
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P. Gmeiner et al.
8.5 Hz, 1H), 4.41 (s, 1H), 6.93 (d, J=8.3 Hz, 1H), 8.15 (d, J=8.3 Hz,
1H); 13C NMR (90 MHz, CD3OD): d=16.1, 24.0, 29.4, 37.0, 39.8, 43.0,
43.1, 48.0, 52.2, 67.1, 70.0, 74.7, 96.3, 108.5, 116.1, 116.9, 126.6,
130.2, 144.5, 154.2, 172.9, 174.1, 174.4, 188.1, 195.6; IR (neat): n˜ =
3500–3200, 2954, 2098, 1673, 1616 cmꢀ1; APCI-MS (m/z): calcd for
C27H33N6O9: 585.2 [M+H]+, found: 585.2 [M+H]+, 568.2
[M+HꢀOH]+.
oil; IR (neat): n˜ =3289, 2935, 1963, 1731, 1666, 1542, 1388,
1099 cmꢀ1
.
(S)-N-(5-Azidopentanoyl)-alanine (5a): The compound was syn-
thesized by using 2-chlorotritylchloride resin (200 mg, 0.32 mmol),
(S)-Fmoc-alanine (99.6 mg, 0.32 mmol), DIPEA (223 mL, 1.28 mmol)
for resin loading and 5-azidopentanoic acid[14] (229.0 mg,
1.60 mmol), PyBOP (832.6 mg, 1.60 mmol), DIPEA (279 mL,
1.60 mmol) and HOBt (324.3 mg, 2.4 mmol) for peptide coupling.
General procedure for the synthesis of N-terminally modified
amino acid building blocks 4a-c and 5a,b: Commercially avail-
able 2-chlorotritylchloride resin (loading: 1.6 mmolgꢀ1) was stirred
in a solution of the corresponding Fmoc-protected amino acid
(1 equiv)/diisopropylethylamine (DIPEA, 4 equiv) in CH2Cl2. After
2 h, the resin was washed with CH2Cl2/CH3OH/DIPEA (17:2:1, 3ꢂ),
CH2Cl2 (3ꢂ), DMF (2ꢂ), and dichloromethane (2ꢂ) and dried in
vacuo. For Fmoc deprotection, the resin was treated with piperi-
dine (20%) in DMF (1ꢂ, microwave irradiation: 5ꢂ5 s, 100 W), fol-
lowed by washings with DMF (5ꢂ). Peptide coupling was done by
employing 5 equiv of the corresponding linker (hexynoic acid or
5-azidopentanoic acid[14])/PyBOP/DIPEA and 7.5 equiv HOBt, dis-
solved in a minimum amount of DMF (irradiation: 15ꢂ10 s, 50 W).
In between each irradiation step, cooling of the reaction mixture
to a temperature of ꢀ108C was achieved by sufficient agitation in
an ethanol–ice bath. Cleavage from the resin was performed by
treatment with hexafluoroisopropanol/dichloromethane (1:4) for
20 min followed by filtration and removal of the solvent in vacuo.
The crude peptides were obtained in very high purities and em-
ployed for the following reactions without further purification.
1
Yield: 65.5 mg (92%), colourless oil; H NMR (600 MHz, CDCl3): d=
1.44 (d, J=7.2 Hz, 3H), 1.62 (tt, J=7.2, 7.4 Hz, 2H), 1.71 (tt, J=7.6,
7.4 Hz, 2H), 2.29 (t, J=7.6 Hz, 2H), 3.29 (t, J=7.2 Hz, 2H), 4.57 (dq,
J=7.2, 7.2 Hz, 1H), 6.57 (d, J=7.2 Hz, 1H), 10.25 (brs, 1H); 13C NMR
(90 MHz, CDCl3): d=18.0, 22.6, 28.1, 35.5, 48.2, 51.0, 173.3, 175.6; IR
(neat): n˜ =3309, 2942, 2098, 1727, 1646, 1542, 1454, 1234 cmꢀ1
.
(S)-N-(5-Azidopentanoyl)-phenylalanine (5b): The compound was
synthesized by using 2-chlorotritylchloride resin (200 mg,
0.32 mmol), (S)-Fmoc-phenylalanine (124.0 mg, 0.32 mmol), DIPEA
(223 mL, 1.28 mmol) for resin loading and 5-azidopentanoic acid[14]
(229.0 mg, 1.60 mmol), PyBOP (832.6 mg, 1.60 mmol), DIPEA
(279 mL, 1.60 mmol) and HOBt (324.3 mg, 2.4 mmol) for peptide
coupling. Yield: 74.0 mg (78%), colourless oil; 1H NMR (600 MHz,
CDCl3): d=1.47–1.69 (m, 4H), 2.20 (m, 2H), 3.10 (dd, J=14.0,
6.7 Hz, 1H), 3.24 (dd, J=14.0, 6.7 Hz, 1H), 3.23 (t, J=6.6 Hz, 2H),
4.87 (ddd, J=7.5, 6.7, 6.7 Hz, 1H), 6.11 (d, J=7.5 Hz, 1H), 7.12–7.32
(m, 5H), 7.50 (brs, 1H); 13C NMR (90 MHz, CDCl3): d=22.6, 28.1,
35.6, 37.3, 51.0, 53.2, 127.2, 128.6, 129.3, 135.8, 173.2, 174.7; IR
(neat): n˜ =3309, 3062, 3031, 2935, 2869, 1731, 1650, 1261,
1099 cmꢀ1; EI-HRMS (m/z) calcd. for C8H11NO3, 290.1379; found,
290.1379.
(S)-N-(5-Hexynoyl)-alanine (4a): The compound was synthesized
by using 2-chlorotritylchloride resin (100 mg, 0.16 mmol), (S)-Fmoc-
alanine (49.8 mg, 0.16 mmol), DIPEA (112 mL, 0.64 mmol) for resin
loading and 5-hexynoic acid (77 mL, 0.80 mmol), PyBOP (416.3 mg,
0.80 mmol), DIPEA (139 mL, 0.80 mmol) and HOBt (162.2 mg,
1.2 mmol) for peptide coupling. Yield: 26.0 mg (89%), colourless
General procedure for the azide–alkyne cycloaddition reaction
of the aminodoxycycline derivatives with modified amino acid
building blocks: The acyldoxycycline derivative (1 or 2, 1 equiv),
copper(I) iodide (13 equiv) and ascorbic acid (7 equiv) were dis-
solved in dry, degassed DMF and the flask charged with N2. To this
solution, the modified amino acid modified building block (4a–c,
5a,b, 2 equiv) dissolved in the minimum amount of dry DMF and
DIPEA (17 equiv) was added. The flask was sonicated for 30 s in
order to improve the dissolving of the catalyst and then micro-
wave irradiation (4ꢂ100 W power, 508C temperature limit) was
performed. Between every cycle the vial was cooled to ꢀ108C in
an acetone/ice bath. The mixture was then diluted with H2O and
lyophilized. The crude mixture was redissolved with THF/HCl; this
permitted the filtration of the copper catalyst. THF was evaporated
in vacuo, the crude product dissolved in acetonitrile and purified
by preparative RP-HPLC.
1
oil; H NMR (360 MHz, CDCl3): d=1.47 (d, J=7.2 Hz, 3H), 1.87 (tt,
J=7.3 Hz, 2H), 2.02 (t, J=2.6 Hz, 2H), 2.28 (dt, J=7.3, 2.6 Hz, 2H),
2.41 (t, J=7.3 Hz, 2H), 4.60 (dq, J=7.2, 7.3 Hz, 1H), 6.55 (d, J=
7.3 Hz, 1H), 8.70 (brs, 1H); 13C NMR (90 MHz, CDCl3): d=17.7, 18.0,
24.0, 34.7, 48.2, 69.4, 83.3, 173.2, 175.8; IR (neat): n˜ =3293, 2938,
2117, 1731, 1643, 1546, 1454, 1214, 1168, 651 cmꢀ1; EI-HRMS (m/z):
calcd. for C9H13NO3, 183.0895; found, 183.0895.
(S)-N-(5-Hexynoyl)-phenylalanine (4b): The compound was syn-
thesized using 2-chlorotritylchloride resin (100 mg, 0.16 mmol), (S)-
Fmoc-phenylalanine (62.0 mg, 0.16 mmol), DIPEA (112 mL,
0.64 mmol) for resin loading and 5-hexynoic acid (77 mL,
0.80 mmol), PyBOP (416.3 mg, 0.80 mmol), DIPEA (139 mL,
0.80 mmol) and HOBt (162.2 mg, 1.2 mmol) for peptide coupling.
1
(S)-2-[5-[4-[4-(Doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-
Yield: 38.0 mg (92%), colourless oil; H NMR (360 MHz, CDCl3): d=
1-yl]-pentanoylamino] propanoic acid (6a):
1
(20 mg,
1.79 (tt, J=7.5, 7.5 Hz, 2H), 1.95 (t, J=2.6 Hz, 2H), 2.12–2.23 (m,
2H), 2.32 (t, J=7.5 Hz, 2H), 3.11 (dd, J=14.0, 6.7 Hz, 1H), 3.24 (dd,
J=14.0, 6.7 Hz, 1H), 4.87 (ddd, J=7.5, 6.7, 6.7 Hz, 1H), 6.15 (d, J=
7.5 Hz, 1H), 7.15–7.32 (m, 5H), 7.90 (brs); 13C NMR (90 MHz, CDCl3):
d=17.7, 24.0, 34.7, 37.4, 53.3, 69.4, 83.3, 127.2, 128.7, 129.4, 135.8,
173.0, 174.6; IR (neat): n˜ =3293, 3031, 2935, 2117, 1731, 1646,
1542, 1438, 1384, 1219, 701, 667 cmꢀ1; EI-HRMS (m/z) calcd. for
C15H17NO3, 259.1208; found, 259.1208.
0.036 mmol), CuI (89.0 mg, 0.47 mmol) and ascorbic acid (44.0 mg,
0.25 mmol), dissolved in DMF (2 mL), were treated with a solution
of 5a (15.4 mg, 0.072 mmol) and DIPEA (100 mL, 0.614 mmol) in
DMF (1 mL) following the general procedure. Purification: P1, tR:
10.6 min. Yield: 5.5 mg (20%), yellow film. Analytical HPLC: tR:
1
14.9 min, purity: >99% (254 nm); H NMR (600 MHz, CD3OD): d=
1.37 (d, 3H, J=7.5 Hz), 1.54 (d, 3H, J=6.8 Hz), 1.70 (tt, 2H, J=7.2,
7.2 Hz), 1.91–2.04 (m, 4H), 2.27 (t, 2H, J=7.2 Hz), 2.51 (t, 2H, J=
7.2 Hz), 2.57 (dd, 1H, J=12.3, 8.6 Hz), 2.71–2.79 (m, 3H, H-6), 2.82
(d, 1H, J=11.3 Hz), 2.96 (brs, 6H), 3.56 (dd, 1H, J=11.3, 8.6 Hz),
4.36 (q, 1H, J=7.5 Hz), 4.39–4.45 (m, 4H, CH2N-N), 6.94 (d, 1H, J=
8.2 Hz), 7.81 (s, 1H), 8.15 (d, 1H, J=8.2 Hz); 13C NMR (90 MHz,
CD3OD): d=16.1, 17.6, 23.6, 25.6, 26.5, 30.5, 35.7, 36.8 39.8, 43.0,
43.1, 48.0, 50.9, 70.1, 70.1, 74.6, 74.6, 96.3, 108.6, 116.2, 117.0, 123.6,
N-(5-Hexynoyl)-glycine (4c): The compound was synthesized by
using 2-chlorotritylchloride resin (138 mg, 0.22 mmol), Fmoc-gly-
cine (65.4 mg, 0.22 mmol), DIPEA (154 mL, 0.88 mmol) for resin
loading and 5-hexynoic acid (121 mL, 1.10 mmol), PyBOP (572.4 mg,
1.10 mmol), DIPEA (192 mL, 1.10 mmol) and HOBt (223.0 mg,
1.65 mmol) for peptide coupling. Yield: 32.0 mg (86%), colourless
708
www.chembiochem.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 703 – 712

Tetracycline–Amino Acid Conjugates
126.7, 130.3, 144.6, 148.4, 154.2, 173.0, 174.0, 174.1, 175.3, 176.1,
188.1, 195.6; IR (neat): n˜ =3500–3200, 2950, 1781, 1670, 1623,
1531, 1430, 1195, 1153 cmꢀ1; APCI-MS (m/z): calcd for C36H46N7O12:
768.3 [M+H]+, found: 768.3 [M+H]+, 751.3 [M+HꢀOH]+.
175.3, 188.1, 195.6; IR (neat): n˜ =3550–3170, 2945, 1737, 1665,
1615, 1529, 1431, 1241, 1033 cmꢀ1; APCI-MS (m/z): calcd for
C42H50N7O12: 844.3 [M+H]+, found: 844.4 [M+H]+, 827.4
[M+HꢀOH]+.
(S)-2-[5-[4-[4-(Doxycyclin-9-ylamino)-4-oxobutyl]-1H-1,2,3-triazol-
1-yl]-pentanoylamino]-3-phenylpropanoic acid (6b): 1 (20 mg,
0.036 mmol), CuI (89.0 mg, 0.47 mmol) and ascorbic acid (44.0 mg,
0.25 mmol), dissolved in DMF (2 mL), were treated with a solution
of 5b (20.9 mg, 0.072 mmol) and DIPEA (100 mL, 0.614 mmol) in
DMF (1 mL) following the general procedure. Purification: P2, tR:
14.4 min. Yield: 5.5 mg (20%), yellow film. Analytical HPLC: tR:
2-[4-[1-[5-(Doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-triazol-4-
yl]-butyrylamino] acetic acid (8c): 2 (20 mg, 0.034 mmol), CuI
(84.0 mg, 0.44 mmol) and ascorbic acid (42.0 mg, 0.24 mmol), dis-
solved in DMF (2 mL), were treated with a solution of 4c (11.5 mg,
0.068 mmol) and DIPEA (100 mL, 0.614 mmol) in DMF (1 mL) follow-
ing the general procedure. Purification: P1, tR: 17.0 min. Yield:
15.4 mg (53%), yellow film. Analytical HPLC (S1A): tR: 14.6 min,
purity: 96% (254 nm); IR (neat): n˜ =3530–3190, 2957, 2878, 1741,
1
16.8 min, purity: >74% (254 nm); H NMR (600 MHz, CD3OD): d=
1672, 1620, 1530, 1428, 1241, 1201, 1138 cmꢀ1; H NMR (600 MHz,
1.48 (tt, J=7.5, 7.5 Hz, 2H), 1.54 (d, J=6.4 Hz, 3H), 1.66–1.79 (m,
2H), 2.07 (tt, J=7.3, 7.2 Hz, 2H), 2.14–2.23 (m, 2H), 2.52 (t, J=
7.2 Hz, 2H), 2.56 (dd, J=12.8, 8.3 Hz, 1H), 2.74 (dq, J=12.8, 6.4 Hz,
1H), 2.80 (t, J=7.3 Hz, 2H), 2.81 (d, J=11.5, 1H), 2.90 (dd, J=14.0,
9.8 Hz, 1H), 2.95 (brs, 6H), 3.21 (dd, J=14.0, 4.5 Hz, 1H), 3.56 (dd,
J=11.5, 8.3 Hz, 1H), 4.28 (t, J=7.0 Hz, 2H), 4.41 (s, 1H), 4.68 (dd,
J=9.8, 4.2 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 7.14–7.25 (m, 5H), 7.72
(s, 1H), 8.14 (d, J=8.3 Hz, 1H); 13C NMR (150 MHz, CD3OD): d=
16.1, 23.5, 25.6, 26.5, 30.3, 35.8, 36.8, 38.4, 39.8, 42.9, 43.0, 48.0,
50.8, 54.8, 67.1, 70.0, 74.7, 96.2, 108.5, 116.1, 116.9, 123.5, 126.6,
127.7, 129.4, 130.2, 130.3, 138.6, 144.5, 148.5, 154.2, 172.9, 174.3,
174.5, 174.9, 175.4, 188.1, 195.6; IR (neat): n˜ =3560–305, 2933,
2876, 1743, 1666, 1614, 1529, 1432, 1241, 1191, 1033 cmꢀ1; APCI-
MS (m/z): calcd for C42H50N7O12: 844.3 [M+H]+, found: 844.4
[M+H]+, 827.4 [M+HꢀOH]+.
1
CD3OD): d=1.54 (d, J=6.8 Hz, 3H), 1.70 (tt, J=7.3, 7.3 Hz, 2H),
1.94–2.03 (m, 4H), 2.29 (t, J=7.4 Hz, 2H), 2.51 (t, J=7.3 Hz, 2H),
2.56 (dd, J=11.9, 8.6 Hz, 1H), 2.74 (t/m, J=7.4 Hz, 3H), 2.79 (d, J=
11.0, 1H), 2.94 (brs, 6H), 3.57 (dd, J=11.0, 8.6 Hz, 1H), 3.87 (s, 2H),
4.38 (s, 1H), 4.43 (t, J=6.8 Hz, 2H), 6.93 (d, J=8.3 Hz, 1H), 7.80 (s,
1H), 8.11 (d, J=8.3 Hz, 1H); APCI-MS (m/z): calcd for C35H44N7O12:
754.3 [M+H]+, found: 754.4 [M+H]+, 737.3 [M+HꢀOH]+.
General procedure for the preparation of methyl ester deriva-
tives from compounds 7a,b and 9a,b: To a solution of the re-
spective carboxylic acid derivative (6a,b and 8a–c) in methanol
(1 mL) concentrated hydrochloric acid (0.1 mL) was added. After
stirring at room temperature for 14 h, H2O (2 mL) was added and
direct purification by preparative RP-HPLC was performed.
(S)-2-[4-[1-[5-(Doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-tria-
(S)-Methyl
2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-
zol-4-yl]-butyrylamino] propanoic acid (8a):
2
(20 mg,
1,2,3-triazol-1-yl]-pentanoylamino]-propanoate (7a): 6a (10 mg,
0.013 mmol) was reacted with MeOH/HCl following the general
procedure. Purification: P1. Yield: 5.5 mg (20%), yellow film. Analyt-
0.034 mmol), CuI (84.0 mg, 0.44 mmol) and ascorbic acid (42.0 mg,
0.24 mmol), dissolved in DMF (2 mL), were treated with a solution
of 4a (12.5 mg, 0.068 mmol) and DIPEA (100 mL, 0.614 mmol) in
DMF (1 mL) following the general procedure. Purification: P1, tR =
13.5 min. Yield: 6.4 mg (24%), yellow film. Analytical HPLC: tR =
1
ical HPLC: tR: 15.2 min, purity: >85% (254 nm); H NMR (600 MHz,
CD3OD): d=1.36 (d, J=7.4 Hz, 3H), 1.55 (d, J=6.6 Hz, 3H), 1.58–
1.65 (m, 2H), 1.96 (tt, J=7.4, 7.4 Hz, 2H), 2.08–2.14 (m, 2H), 2.25–
2.32 (m, 2H), 2.55 (t, J=7.3 Hz, 2H), 2.59 (dd, J=12.8, 8.3 Hz, 1H),
2.76 (dq, J=12.8, 6.6 Hz, 1H), 2.83 (d, J=11.5 Hz, 1H), 2.85–2.89
(m, 2H), 2.96 (brs, 6H), 3.57 (dd, J=11.5, 8.3 Hz, 1H), 3.69 (s, 3H),
4.37 (q, J=7.4 Hz, 1H), 4.43 (s, 1H), 4.45 (t, J=7.4 Hz, 2H), 6.95 (d,
J=8.3 Hz, 1H), 8.04 (s, 1H), 8.14 (d, J=8.3 Hz, 1H); IR (neat): n˜ =
3500–3120, 3066, 2952, 2861, 1734, 1667, 1634, 1597, 1550, 1437,
1244, 1195, 1043 cmꢀ1; APCI-MS (m/z): calcd for C37H48N7O12: 782.3
[M+H]+, found: 782.4 [M+H]+, 765.4 [M+HꢀOH]+.
1
14.9 min, purity: >99% (254 nm); H NMR (600 MHz, CD3OD): d=
1.37 (d, J=7.2 Hz, 3H), 1.54 (d, J=6.8 Hz, 3H), 1.57–1.67 (m, 2H),
1.88–1.98 (m, 2H), 2.01–2.13 (m, 2H), 2.27 (t, J=7.4 Hz, 2H), 2.51
(brs, 2H), 2.57 (dd, J=12.5, 8.3 Hz, 1H), 2.71–2.78 (m, 3H), 2.82 (d,
J=11.4 Hz, 1H), 2.95 (brs, 6H), 3.56 (dd, J=11.4, 8.5 Hz, 1H), 4.30–
4.45 (m, 4H), 6.94 (d, J=8.3 Hz, 1H), 7.80 (s, 1H), 8.13 (d, J=8.3 Hz,
1H); IR (neat): n˜ =3500–3200, 2942, 1781, 1674, 1619, 1531, 1427,
1195, 1137, 1037 cmꢀ1; APCI-MS (m/z): calcd for C36H46N7O12: 768.3
[M+H]+, found: 768.3 [M+H]+, 751.3 [M+H+OH]+.
(S)-2-[5-[4-[4-(Doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-tria-
zol-1-yl]-butyrylamino]-3-phenylpropanoic acid (8b): 2 (20 mg,
0.034 mmol), CuI (84.0 mg, 0.44 mmol) and ascorbic acid (42.0 mg,
0.24 mmol), dissolved in DMF (2 mL), were treated with a solution
of 4b (17.6 mg, 0.068 mmol) and DIPEA (100 mL, 0.614 mmol) in
DMF (1 mL) following the general procedure. Purification: P2, tR:
15.3 min. Yield: 5.5 mg (20%), yellow film. Analytical HPLC: tR =
16.8 min, purity: 94% (254 nm); 1H NMR (600 MHz, CD3OD): d=
1.53 (d, J=6.8 Hz, 3H), 1.69 (tt, J=7.3, 7.2 Hz, 2H), 1.85 (tt, J=7.4,
7.5 Hz, 2H), 2.00 (tt, J=7.3, 6.7 Hz, 2H), 2.17–2.22 (m, 2H), 2.51 (t,
J=7.2 Hz, 2H), 2.56 (dd, J=13.0, 8.5 Hz, 1H), 2.58 (m, 2H), 2.72
(dq, J=13.0, 6.8 Hz, 1H), 2.82 (d, 1H), 2.90 (1H, 9.5 Hz, J=14.2),
2.95 (brs, 6H), 3.21 (dd, J=14.2, 4.9 Hz, 2H), 3.56 (dd, J=11.3,
8.5 Hz, 1H), 4.41 (s, 1H), 4.42 (t, J=6.7 Hz, 2H), 4.68 (dd, J=9.5,
4.9 Hz, 1H), 6.91 (d, J=8.3 Hz, 1H), 7.14–7.25 (m, 5H), 7.71 (s, 1H),
8.12 (d, J=8.3 Hz, 1H); 13C NMR (90 MHz, CD3OD): d=16.1, 23.7,
25.4, 26.5, 30.7, 35.8, 36.7, 38.4, 39.8, 43.1, 43.2, 48.0, 50.8, 54.8,
67.1, 70.0, 74.6, 96.2, 108.4, 116.2, 116.9, 123.4, 126.5, 127.7, 129.4,
130.2, 130.37, 138.5, 144.6, 148.5, 154.21, 172.9, 174.2, 174.4, 174.8,
(S)-Methyl
2-[5-[4-[4-(doxycyclin-9-ylamino)-4-oxobutyl]-1H-
1,2,3-triazol-1-yl]-pentanoylamino]-3-phenylpropanoate (7b): 6b
(10 mg, 0.012 mmol) was reacted with MeOH/HCl following the
general procedure. Purification: P1, tR: 20.9 min. Yield: 9.2 mg
(91%), yellow film. Analytical HPLC: tR =17.6 min, purity: 93%
(254 nm); IR (neat): n˜ =3500–3200, 3061, 2927, 2873, 1739, 1662,
1616, 1527, 1430, 1241, 1195, 1033 cmꢀ1
;
1H NMR (600 MHz,
CD3OD): d=1.48 (tt, J=7.4, 7.4 Hz, 2H), 1.54 (d, J=6.5 Hz, 3H),
1.68–1.79 (m, 2H), 2.08 (tt, J=7.2, 7.2 Hz, 2H), 2.14–2.23 (m, 2H),
2.52 (t, J=7.2 Hz, 2H), 2.57 (dd, J=12.8, 8.4 Hz, 1H), 2.74 (dq, J=
12.8, 6.5 Hz, 1H), 2.80 (t J=7.2 Hz, 2H), 2.81 (d, J=11.4, 1H), 2.90
(dd, J=14.0, 9.8 Hz, 1H), 2.95 (brs, 6H), 3.21 (dd, J=14.0, 5.2 Hz,
1H), 3.56 (dd, J=11.4, 8.4 Hz, 1H), 3.67 (s, 3H), 4.30 (t, J=7.0 Hz,
2H), 4.42 (s, 1H), 4.67 (dd, J=9.8, 5.2 Hz, 1H), 6.94 (d, J=8.3 Hz,
1H), 7.14–7.25 (m, 5H), 7.75 (s, 1H), 8.15 (d, J=8.3 Hz, 1H);
13C NMR (150 MHz, CD3OD): d=16.1, 23.5, 25.6, 26.5, 30.4, 35.7,
36.8, 38.4, 39.8, 43.0, 48.0, 50.9, 52.7, 55.1, 67.1, 70.0, 74.6, 96.2,
108.5, 116.2, 116.9, 123.5, 126.6, 127.9, 129.5, 130.2, 130.3, 138.3,
144.5, 148.4, 154.2, 172.9, 173.6, 174.1, 174.3, 175.3, 188.2, 195.6;
ChemBioChem 2010, 11, 703 – 712
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
709

P. Gmeiner et al.
APCI-MS (m/z): calcd for C43H52N7O12: 858.4 [M+H]+, found: 858.4
[M+H]+, 841.3 [M+HꢀOH]+.
d=29.8, 37.5, 46.0, 50.2, 63.6, 173.0; IR (neat): n˜ =3350–3200, 2969,
2942, 2869, 2823, 2780, 2098, 1666, 1527, 1457, 1265, 1045 cmꢀ1
.
EI-HRMS (m/z) calcd. for C7H15N5O, 185.1277; found, 185.1278.
(S)-Methyl
2-[4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-
1,2,3-triazol-4-yl]-butyrylylamino]-3-phenylpropanoate (9a): 8b
(10 mg, 0.012 mmol) was reacted with MeOH/HCl following the
general procedure. Purification: P1, tR =19.0 min. Yield: 6.1 mg
(60%), yellow film. Analytical HPLC: tR =17.1 min, purity: 99%
(254 nm); 1H NMR (600 MHz, CD3OD): d=1.53 (d, J=6.4 Hz, 3H),
1.65–1.73 (m, 2H), 1.82–1.89 (m, 2H), 1.95–2.04 (m, 2H), 2.15–2.24
(m, 2H), 2.48–2.53 (m, 2H), 2.53–2.63 (m, 3H), 2.70–2.76 (m, 1H),
2.81 (d, J=11.2 Hz, 1H), 2.91 (dd, J=14.0, 9.4 Hz, 1H), 2.98 (brs,
6H), 3.21 (dd, J=14.0, 5.8 Hz, 1H), 3.56 (dd, J=11.2, 8.3 Hz, 1H),
3.68 (s, 3H), 4.40–4.44 (m, 3H), 4.67 (dd, J=9.4, 5.8 Hz, 1H), 6.93
(d, J=8.3 Hz, 1H), 7.15–7.27 (m, 5H), 7.72 (s, 1H), 8.12 (d, J=
8.3 Hz, 1H); IR (neat): n˜ =3600–3160, 3063, 2977, 2955, 2874, 1732,
1670, 1615, 1529, 1428, 1241, 1199, 1045 cmꢀ1; APCI-MS (m/z):
calcd for C43H52N7O12: 858.4 [M+H]+, found: 858.4 [M+H]+, 841.4
[M+HꢀOH]+.
(S)-2-Amino-N-(3-azidopropyl)-propanamide (11b2): The reaction
was carried out employing Boc-l-alanine (832.0 mg). Flash chroma-
tography: petroleum ether/ethyl acetate (4:1). Yield of the Boc de-
rivative 11 b1: 420.0 mg (77%), yellow oil, which was deprotected
1
following the general procedure to give 11 b2. H NMR (600 MHz,
CDCl3) of 11 b1 d 1.27 (d, J=7.0 Hz, 3H), 1.36 (s, 9H), 1.71 (tt, J=
6.7, 6.7 Hz, 2H), 3.23 and 3.27 (2ꢂ t, each 2H, each J=6.7 Hz), 4.10
(brs, 1H), 5.44, 6.95 (2ꢂbrs, each 1H); 13C NMR (90 MHz, CD3OD):
d=18.4, 28.3, 28.6, 36.6, 48.9, 50.0, 79.6, 155.4, 173.1; IR (neat): n˜ =
of 11 b1 3450–3150, 2978, 2933, 2874, 2509, 2096, 1681, 1651,
1455, 1368, 1248, 1167, 1056, 1020 cmꢀ1
.
(S)-2-Amino-N-(3-azidopropyl)-3-phenyl-propanamide
(11c2):
The reaction was carried out by employing Boc-l-phenylalanine
(1167.0 mg). Flash chromatography: petroleum ether/ethyl acetate
(4:1). Yield of the Boc derivative 11 c1: 695.0 mg (100%), yellow oil,
which was deprotected following the general procedure to give
Methyl 2-[4-[1-[5-(doxycyclin-9-ylamino)-5-oxopentyl]-1H-1,2,3-
triazol-4-yl]-butyrylylamino]-acetate
(9b):
8c
(10 mg,
1
11 c2; H NMR (360 MHz, CDCl3) of 11 c1: d=1.41 (s, 9H), 1.63 (tt,
0.013 mmol) was reacted with MeOH/HCl following the general
procedure. Purification: P1, tR =11.0 min. Yield: 5.4 mg (53%),
yellow film. Analytical HPLC: tR =14.9 min, purity: >90% (254 nm);
1H NMR (600 MHz, CD3OD): d=1.54 (d, J=6.8 Hz, 3H), 1.70 (tt, J=
7.5, 7.3 Hz, 2H), 1.94–2.04 (m, 4H), 2.29 (t, J=7.4 Hz, 2H), 2.51 (t,
J=7.3 Hz, 2H), 2.57 (dd, J=12.4, 8.3 Hz, 1H), 2.69–2.77 (m, 1H),
2.75 (t, J=7.4 Hz, 2H), 2.82 (d, J=11.5, 1H), 2.96 (brs, 6H), 3.56 (dd,
J=11.5, 8.3 Hz, 1H), 3.70 (s, 3H), 3.89 (s, 2H), 4.43 (t, J=6.9 Hz,
2H), 6.93 (d, J=8.3 Hz, 1H), 7.81 (s, 1H), 8.11 (d, J=8.3 Hz, 1H); IR
(neat): n˜ =3500–3200, 3081, 2954, 2873, 1751, 1670, 1616, 1527,
1430, 1241, 1195, 1141 cmꢀ1; APCI-MS (m/z): calcd for C36H46N7O12:
768.3 [M+H]+, found: 768.4 [M+H]+, 751.4 [M+HꢀOH]+.
J=6.6, 6.6 Hz, 2H), 3.00–3.34 (m, 6H), 4.27–4.36 (m, 1H), 5.23 (d,
J=7.9 Hz, 1H), 6.25 (t, J=4.9 Hz, 1H), 7.15–7.32 (m, 5H); 13C NMR
(90 MHz, CD3OD): d=28.2, 28.5, 36.8, 38.7, 48.9, 56.1, 80.1, 126.9,
128.6, 129.2, 136.7, 155.4, 171.4; IR (neat): n˜ =of 11 c1: 3309, 3062,
3031, 2935, 2869, 2098, 1666, 1527, 1457, 1265, 1045 cmꢀ1. EI-
HRMS (m/z)of 11 c1 calcd. for C17H25N5O3, 347.1957; found,
347.1957.
(S)-2-Amino-N-(2-(3-azidopropylamino)-2-oxoethyl)-propanamide
(11d2): The reaction was carried out by employing Boc-Ala-Gly-OH
(591.0 mg, 2.4 mmol), EDC (0.43 mL, 2.4 mmol), triethylamine
(0.46 mL, 3.27 mmol) and HOAt (321.8 mg, 2.4 mmol). Washing
steps of the ethyl acetate layer with 1n HCl, brine and 1n NaOH
were sufficient to obtain a pure peptide. Yield of the Boc derivative
11 d1: 656.0 mg (100%, crude), light yellow oil, which was depro-
tected following the general procedure to give 11 d2; IR (neat): n˜ =
of 11 d1: 3600–3200, 2946, 2877, 2827, 2780, 2098, 1666, 1527,
General procedure for the synthesis of C-terminally modified
amino acid building blocks 11a,b2,c2,d2: The corresponding
amino acid (4.4 mmol), N-(3-dimethylaminopropyl)-N’-ethyl-carbo-
diimide (EDC, 0.78 mL, 4.4 mmol) and triethylamine (0.84 mL,
6 mmol) were dissolved in dry DMF (10 mL) and cooled to 08C.
Thereafter, a solution of HOAt (598.9 mg, 4.4 mmol) in DMF (5 mL)
1
1457, 1268, 1045 cmꢀ1; H NMR (600 MHz, [D6]DMSO) of 11 d1: d=
solution of 3-amino-1-azidopropane[20]
1.17 (d, J=7.2 Hz, 3H), 1.39 (s, 9H), 1.66 (tt, J=6.8, 6.8 Hz, 2H),
3.05–3.13 and 3.13–3.20 (2ꢂm, 2H), 3.34 (t, J=6.8 Hz, 2H), 3.61
(dd, J=16.4, 5.5 Hz, 1H), 3.67 (dd, J=16.4, 5.5 Hz, 1H), 3.92 (dq,
J=7.2, 6.6 Hz, 1H), 7.09 (d, J=6.6 Hz, 1H), 7.70 (dd, J=5.5, 5.5 Hz,
1H), 8.06–8.12 (brs, 1H); 13C NMR (90 MHz, [D6]DMSO) of 11 d1:
d=17.6, 28.2, 28.3, 35.8, 42.2, 48.3, 50.0, 78.3, 155.4, 168.74, 173.0.
and after 5 min,
a
(200.2 mg, 2 mmol) in DMF (1.5 mL) were added. After stirring for
30 min, the mixture was allowed to warm to RT. After 14 h, aque-
ous citric acid (10%, 200 mL, in case of 11 a 1 n NaOH) was added
and extraction with ethyl acetate (4ꢂ100 mL) was performed. The
combined organic layers were washed with HCl (1 n, 1ꢂ, except
for 11 a, this step was omitted), brine (1ꢂ) and 1 n NaOH (2ꢂ),
dried over Na2SO4 and concentrated in vacuo. Purification by flash
chromatography on silica gel afforded the pure amino acid deriva-
tive 11 a and the intermediates 11 b1–d1. In the case of 11 b1–d1,
Boc-deprotection was carried out by using TFA/dichloromethane
(1:1, 5 mL) and stirring at room temperature for 1 h. Solvent was
removed in vacuo, then sat. aqueous NaHCO3 (10 mL) was added
and the solution extracted with ethyl acetate (3ꢂ). The combined
organic layers were dried over Na2SO4 and evaporated in vacuo to
afford the amino acid derivatives 11 b2–d2, which were used with-
out further purification.
9-[4-[1-[3-[2-(Dimethylamino)-acetamido]propyl]-1H-1,2,3-triazol-
4-yl]butyrylamino]doxycycline (12a): 1 (25 mg, 0.045 mmol), CuI
(111.0 mg, 0.58 mmol) and ascorbic acid (55.0 mg, 0.32 mmol), dis-
solved in DMF (2 mL), were treated with a solution of 11 a
(17.0 mg, 0.092 mmol) and DIPEA (131 mL, 0.614 mmol) in DMF
(1 mL) following the general procedure. Purification: P2, tR: 8.7 min.
Yield: 5.6 mg (17%), yellow film. Analytical HPLC: tR =3.1 min,
1
purity: >95% (254 nm); H NMR (600 MHz, CD3OD): d=1.57 (d, J=
6.7 Hz, 3H), 2.12 (tt, J=7.5, 7.4 Hz, 2H), 2.19 (tt, J=6.9, 6.9 Hz, 2H),
2.58 (t, J=7.4 Hz, 2H), 2.61 (dd, J=12.8, 8.4 Hz, 1H), 2.79 (dq, J=
12.8, 6.7 Hz, 1H), 2.85 (d, J=11.3 Hz, 1H), 2.91 (t J=7.5 Hz, 2H,),
2.92–3.04 (m, 12H), 3.31 (m, 2H), 3.58 (dd, J=11.3, 8.4 Hz, 1H),
3.97 (s, 2H), 4.45 (s, 1H), 4.54 (t, J=6.9 Hz, 2H), 6.97 (d, J=8.3 Hz,
1H), 8.14 (s, 1H), 8.16 (d, J=8.3 Hz, 1H); IR (neat): n˜ =3550–3200,
3047, 2971, 2859, 1669, 1622, 1521, 1471, 1430, 1204, 1129 cmꢀ1
;
APCI-MS (m/z): calcd for C35H47N8O10: 739.3 [M+H]+, found: 739.4
[M+H]+, 722.3 [M+HꢀOH]+.
N-(3-Azidopropyl)-2-(dimethylamino)-acetamide (11a): The reac-
tion was carried out employing N,N-dimethylglycine (453.7 mg).
Flash chromatography: petroleum ether/ethyl acetate 1:1. Yield:
1
362 mg (98%), yellow oil; H NMR (600 MHz, CD3OD): d=1.77 (tt,
J=6.7, 6.7 Hz, 2H), 2.29 (s, 6H), 2.96 (s, 2H), 3.35 and 3.39 (2ꢂt,
each 2H, each J=6.7 Hz), 4.84 (s, 1H); 13C NMR (90 MHz, CD3OD):
710
www.chembiochem.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 703 – 712

Tetracycline–Amino Acid Conjugates
9-[4-[1-[3-((S)-2-Aminopropionylamino)-propyl]-1H-1,2,3-triazol-
4-yl]-butyrylamino] doxycycline (12b): 1 (20 mg, 0.036 mmol), CuI
(89.0 mg, 0.47 mmol) and ascorbic acid (44.0 mg, 0.25 mmol), dis-
solved in DMF (2 mL), were treated with a solution of 11 b2
(12.0 mg, 0.072 mmol) and DIPEA (100 mL, 0.614 mmol) in DMF
(1 mL) following the general procedure. Purification: P1, tR =
9.7 min. Yield: 8.1 mg (31%), yellow film. Analytical HPLC: tR =
12.5 min, purity: 86% (254 nm); 1H NMR (600 MHz, CD3OD): d=
1.50 (d, J=7.1 Hz, 3H), 1.55 (d, J=6.7 Hz, 3H), 2.07 (tt, J=7.4,
7.3 Hz, 2H), 2.13 (tt, J=7.0, 7.0 Hz, 2H), 2.53 (t, J=7.3 Hz, 2H), 2.60
(dd, J=12.9, 8.3 Hz, 1H), 2.76 (dq, J=12.9, 6.7 Hz, 1H), 2.82 (t, J=
7.4 Hz, 2H), 2.84 (d, J=11.5 Hz, 1H), 2.91–3.02 (brs), 3.20–3.27 (m,
2H), 3.57 (dd, J=11.5, 8.3 Hz, 1H), 3.91 (q, J=7.1 Hz, 1H), 4.43 (t,
J=7.0 Hz, 2H), 4.44 (s, 1H), 6.95 (d, J=8.3 Hz, 1H), 7.86 (s, 1H),
8.15 (d, J=8.3 Hz, 1H); IR (neat): n˜ =3500–3190, 3052, 2971, 2920,
2859, 1673, 1622, 1523, 1471, 1430, 1202, 1135 cmꢀ1; APCI-MS
(m/z): calcd for C34H45N8O10: 725.3 [M+H]+, found: 725.4 [M+H]+,
708.4 [M+HꢀOH]+.
galactosidase activities obtained in strains without tetR was set to
100%.
Determination of minimal inhibitory concentration (MIC): For
the determination of the MIC the described method[29] was modi-
fied for measurements in 96-well plates. Each well contained 105
cells of Escherichia coli WH207ltet50,[21] E. coli NR698ltet50[6] or
Bacillus subtilis 168. Each compound was tested at the following
concentrations: 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0 mgLꢀ1. The
cells were incubated in a steam saturated bag at 378C for 18–24 h
on a shaker in the dark. The MIC value represents the lowest con-
centration of a compound at which no growth was detected.
Acknowledgements
This work was supported by the Deutsche Forschungsgemein-
schaft (DFG-SFB473). Iris Torres and Michaela Kettler are ac-
knowledged for skillful technical assistance. We thank Rene Pfei-
fle und Janine Arlt for assistance with some experiments.
9-[4-[1-[3-((S)-2-Amino-3-phenylpropionylamido)propyl]-1H-
1,2,3-triazol-4-yl)butyrylamino]doxycycline (12c):
1
(20 mg,
0.036 mmol), CuI (89.0 mg, 0.47 mmol) and ascorbic acid (44.0 mg,
0.25 mmol), dissolved in DMF (2 mL), were treated with a solution
of 11 c2 (18.0 mg, 0.072 mmol) and DIPEA (100 mL, 0.614 mmol) in
DMF (1 mL) following the general procedure. Purification: P1, tR =
10.9 min. Yield: 15.4 mg (53%), yellow film. Analytical HPLC: tR =
Keywords: antibiotics · click chemistry · doxycycline · reverse
Tet repressor · selective corepressor
[1] R. Tedesco, J. A. Thomas, B. S. Katzenellenbogen, J. A. Katzenellenbogen,
Chem. Biol. 2001, 8, 277–287; D. F. Doyle, D. A. Braasch, L. K. Jackson,
H. E. Weiss, M. F. Boehm, D. J. Mangelsdorf, D. R. Corey, J. Am. Chem.
Soc. 2001, 123, 11367–11371.
[2] a) Y. Pei, S. C. Rogan, F. Yan, B. L. Roth, Physiology 2008, 23, 313–321;
b) K. Scearce-Levie, P. Coward, C. H. Redfern, B. R. Conklin, Trends Phar-
macol. Sci. 2001, 22, 414–420.
1
14.0 min, purity: >90% (254 nm); H NMR (600 MHz, CD3OD): d=
1.55 (d, J=6.8 Hz, 3H), 1.92–2.02 (m, 2H), 2.03–2.10 (m, 2H), 2.50–
2.56 (m, 2H), 2.59 (dd, J=12.4, 8.4 Hz, 1H), 2.72–2.77 (m, 1H),
2.78–2.84 (m, 3H), 2.96 (brs, 6H), 3.07–3.25 (m, 4H), 3.54–3.60 (m,
1H), 4.02–4-.06 (m, 1H), 4.19–4.28 (m, 2H), 4.43 (s, 1H), 6.94 (d, J=
8.2 Hz, 1H), 7.26–7.37 (m, 5H), 7.78 (s, 1H), 8.15 (d, J=8.2 Hz, 1H);
IR (neat): n˜ =3500–3200, 3093, 2992, 2873, 1781, 1670, 1535, 1430,
1168, 1037, 701 cmꢀ1; APCI-MS (m/z): calcd for C40H49N8O10: 801.4
[M+H]+, found: 801.4 [M+H]+, 784.4 [M+HꢀOH]+.
[3] N. Tschammer, M. Doerfler, H. Huebner, P. Gmeiner, ACS Chem. Neurosci.
2009, DOI: 10.1021/cn900001b.
[4] a) C. Denison, T. Kodadek, Chem. Biol. 1998, 5, R129-R145; b) M. Padi-
dam, Curr. Opin. Plant. Biol. 2003, 6, 169–177; c) S. Giuriato, K. Rabin,
A. C. Fan, C. M. Shachaf, D. W. Felsher, Semin. Cancer Biol. 2004, 14, 3–
11; d) C. Toniatti, H. Bujard, R. Cortese, G. Ciliberto, Gene Ther. 2004, 11,
649–657; for a very recent example, see: e) S. B. Cambridge, D. Geissler,
S. Keller, B. Cꢀrten, Angew. Chem. 2006, 118, 2287–2289; Angew. Chem.
Int. Ed. 2006, 45, 2229–2231.
[5] E. M. Henssler, O. Scholz, S. Lochner, P. Gmeiner, W. Hillen, Biochemistry
2004, 43, 9512–9518.
[6] C. Kormann, I. Pimenta, S. Lçber, C. Wimmer, H. Lanig, T. Clark, W. Hillen,
P. Gmeiner, ChemBioChem 2009, 10, 2924–2933.
[7] M. Gossen, S. Freundlieb, G. Bender, G. Mꢀller, W. Hillen, H. Bujard, Sci-
ence 1995, 268, 1766–1769.
[8] C. Berens, S. Lochner, S. Lçber, I. Usai, A. Schmidt, L. Drueppel, W.
Hillen, P. Gmeiner, ChemBioChem 2006, 7, 1320–1324.
[9] a) P.-E. Sum, P. Peterson, Bioorg. Med. Chem. Lett. 1999, 9, 1459–1462;
b) P. Orth, D. Schnappinger, P.-E. Sum, G. A Ellestad, W. Hillen, W. Saeng-
er, W. Hinrichs, J. Mol. Biol. 1999, 285, 455–461.
[10] M. Klotzsche, C. Behrens, W. Hillen, J. Biol. Chem. 2005, 280, 24591–
24599.
[11] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056–
2075; Angew. Chem. Int. Ed. 2001, 40, 2004–2021; b) V. V. Rostovtsev,
L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. 2002, 114, 2708–
2711; Angew. Chem. Int. Ed. 2002, 41, 2596–2599; c) A. Paul, H. Bitter-
mann, P. Gmeiner, Tetrahedron 2006, 62, 8919–8927; d) S. Maschauer, J.
Einsiedel, R. Haubner, C. Hocke, M. Ocker, H. Hꢀbner, T. Kuwert, P.
Gmeiner, O. Prante, Angew. Chem. 2010, 122, 988–992; Angew. Chem.
Int. Ed. 2010, 49, 976–979; e) D. Huber, H. Hꢀbner, P. Gmeiner, J. Med.
Chem. 2009, 52, 6860–6870.
[12] C. Krueger, C. Danke, K. Pfleiderer, W. Schuh, H.-M. Jꢃck, S. Lochner, P.
Gmeiner, W. Hillen, C. Berens, J. Gene Med. 2006, 8, 1037–1047.
[13] T. C. Barden, B. L. Buckwalter, R. T. Testa, P. J. Petersen, V. J. Lee, J. Med.
Chem. 1994, 37, 3205–3211.
9-[4-[1-[3-[2-((S)-2-aminopropionylamino)acetamido]propyl]-1H-
1,2,3-triazol-4-yl]butyrylamino]doxycycline (12d):
1
(20 mg,
0.036 mmol), CuI (89.0 mg, 0.47 mmol) and ascorbic acid (44.0 mg,
0.25 mmol), dissolved in DMF (2 mL), were treated with a solution
of 11 d2 (16.0 mg, 0.072 mmol) and DIPEA (100 mL, 0.614 mmol) in
DMF (1 mL) following the general procedure. Purification: P1, tR =
13.1 min. Yield: 6.2 mg (22%), yellow film. Analytical HPLC: tR =
12.5 min, purity: 90% (254 nm); 1H NMR (600 MHz, CD3OD): d=
1.52 (d, J=7.2 Hz, 3H), 1.55 (d, J=6.5 Hz, 3H), 2.05–2.15 (m, 4H),
2.54 (t, J=7.2 Hz, 2H), 2.59 (dd, J=12.3, 8.3 Hz, 1H), 2.76 (dq, J=
12.3, 6.5 Hz, 1H), 2.81–2.86 (m, 3H), 2.91–3.00 (brs, 6H), 3.24 (t, J=
6.6 Hz, 2H), 3.57 (dd, J=11.3, 8.3 Hz, 1H), 3.84 (d, J=16.6 Hz, 1H),
3.94 (d, J=16.6 Hz, 1H), 4.00 (q, J=7.2 Hz, 1H), 4.43 (s, 1H), 4.44 (t,
J=7.0 Hz, 2H), 6.95 (d, J=8.3 Hz, 1H), 7.90 (s, 1H), 8.15 (d, J=
8.3 Hz, 1H); IR (neat): n˜ =3081, 2981, 2892, 1785, 1650, 1419, 1234,
1172, 1037, 705 cmꢀ1; APCI-MS (m/z): calcd for C36H48N9O11: 782.3
[M+H]+, found: 782.4 [M+H]+, 765.4 [M+HꢀOH]+.
Quantification of TetR induction: Repression and induction, were
determined in E. coli WH207ltet50[21] or E. coli NR698ltet50[6] trans-
formed with tetR variants located on pWH1925.[26] Both E. coli
strains have a chromosomally integrated tetA–lacZ fusion under
control of the tet region from Tn10 so that TetR represses lacZ ex-
pression. Overnight and log phase cultures were grown at 378C in
LB supplemented with ampicillin (100 mgLꢀ1) in absence and pres-
ence of 1.0 mm of the doxycycline derivatives. The b-galactosidase
activities were determined from log phase cultures of three inde-
pendent clones at 378C in 96-well plates as described.[27,28] The b-
[14] N. Khoukhi, M. Vaultier, R. S. Carriꢄ, Tetrahedron 1987, 43, 1811–1822.
ChemBioChem 2010, 11, 703 – 712
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
711

P. Gmeiner et al.
[15] M. A. Kaplan, J. H. Lannon, F. H. Buckwalter, J. Pharm. Sci. 1965, 54, 163–
164.
[24] E. M. Henssler, O. Scholz, S. Lochner, P. Gmeiner, W. Hillen, Biochemistry
2004, 43, 9512–9518.
[16] C. M. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057–
3064.
[25] M. Klotzsche, S. Ehrt, D. Schnappinger, Nucleic Acids Res. 2009, 37,
1778–1788).
[17] H. Jang, J. M. Holub, K. Kirshenbaum, Org. Lett. 2005, 7, 1951–1954.
[18] D. T. S. Rijkers, G. W. van Esse, R. Merkx, A. J. Brouwer, H. J. F. Jacobs, R. J.
Pieters, R. M. J. Liskamp, Chem. Commun. 2005, 4581–4583.
[19] J. Marik, J. L. Sutcliffe, Tetrahedron Lett. 2006, 47, 6681–6684.
[20] N. S. Hatzakis, H. Engelkamp, K. Velonia, J. Hofkens, P. C. M. Christianen,
A. Svendsen, S. A. Patkar, J. Vind, J. C. Maan, A. E. Rowan, R. J. M. Nolte,
Chem. Commun. 2006, 2012–2014.
[26] O. Scholz, E. M. Henssler, J. Bail, P. Schubert, J. Bogdanska-Urbaniak, S.
Sopp, M. Reich, S. Wisshak, M. Kçstner, R. Bertram, W. Hillen, Mol. Micro-
biol. 2004, 53, 777–789.
[27] J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Labo-
ratory Press, New York, 1972.
[28] K. L. Griffith, R. E. Wolf Jr. , Biochem Biophys Res. Commun. 2002, 290,
397–402.
[21] A. Wissmann, L. V. Wray Jr, U. Somaggio, R. Baumeister, M. Geisendorfer,
W. Hillen, Genetics 1991, 128, 225–232.
[29] F. Burkhardt, Mikrobiologische Diagnostik, Thieme, Stuttgart, 1992, 714–
733.
[22] a) H. Nikaido, Chem. Biol. 2005, 12, 507–509; b) N. Ruiz, B. Falcone, D.
Kahne, T. J. Silhavy, Cell 2005, 121, 307–317; c) N. Ruiz, D. Kahne, T. J.
Silhavy, Nat. Rev. Microbiol. 2006, 4, 57–66.
Received: November 19, 2009
[23] O. Scholz, M. Kçstner, M. Reich, S. Gastiger, W. Hillen, J. Mol. Biol. 2003,
329, 217–227.
Published online on February 10, 2010
712
www.chembiochem.org
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ChemBioChem 2010, 11, 703 – 712