Convenient synthesis of multifunctional EDTA-based chiral metal chelates substituted with an S-mesylcysteine

Article abstract of DOI:10.1002/chem.200400907

We describe the synthetic route to ethylenediaminetetraacetic acid (EDTA) derivatives that can be attached to surface-exposed thiol functional groups of cysteine residues in proteins, via a methylthiosulfonate moiety that is connected in a stereochemically unique way to the C-1 carbon atom of EDTA. Such compounds can be used to align proteins in solution without the need to add liquid crystalline media, and are, therefore, of great interest for the NMR spectroscopic analysis of biomolecules. The binding constant for the paramagnetic tag to lanthanide ions was determined by measuring luminescence. For the Tb⁺³-ligand complex, a K_b value of 6.5×10^17-1 was obtained. This value is in excellent agreement with literature values for the related EDTA compound. In addition, it could be shown that there is no significant reduction in the luminescence intensity upon addition of a 10⁴ excess of Ca²⁺ ions, indicating that this paramagnetic tag is compatible with buffers containing high concentrations of divalent alkaline earth ions.

Full text of DOI:10.1002/chem.200400907

Convenient Synthesis of Multifunctional EDTA-Based Chiral Metal Chelates
Substituted with an S-Mesylcysteine
Andrei Leonov,[a] Brigitte Voigt,[a] Fernando Rodriguez-CastaÇeda,[a]
Peyman Sakhaii,[b] and Christian Griesinger*[a]
Abstract: We describe the synthetic
route to ethylenediaminetetraacetic
acid (EDTA) derivatives that can be
attached to surface-exposed thiol func-
tional groups of cysteine residues in
great interest for the NMR spectro-
scopic analysis of biomolecules. The
binding constant for the paramagnetic
tag to lanthanide ions was determined
by measuring luminescence. For the
Tb+3–ligand complex, a Kb value of
6.5ꢀ1017 mÀ1 was obtained. This value is
in excellent agreement with literature
values for the related EDTA com-
pound. In addition, it could be shown
that there is no significant reduction in
the luminescence intensity upon addi-
tion of a 104 excess of Ca2+ ions, indi-
cating that this paramagnetic tag is
compatible with buffers containing
high concentrations of divalent alkaline
earth ions.
proteins, via
a
methylthiosulfonate
moiety that is connected in a stereo-
chemically unique way to the C-1
carbon atom of EDTA. Such com-
pounds can be used to align proteins in
solution without the need to add liquid
crystalline media, and are, therefore, of
Keywords: EDTA · lanthanides ·
NMR spectroscopy
·
protein
tagging · synthetic methods
Introduction
DOTA, DTPA, and their derivatives,[7] has found extensive
use in medicine.
The ability of ethylenediaminetetraacetic acid (EDTA) and
its derivatives to form stable complexes with a variety of
metal ions allows for their use as probes in various areas of
chemistry, biology, and medicine. For example, derivatives
of EDTA–CeIV are effective cleavage reagents for oligonu-
cleotides[1] and are, therefore, used to investigate oligonu-
cleotide structure and function. Protein and nucleic acid
cleavage experiments have been used to probe the tertiary
structure of biomolecules and drug binding sites, as well as
to identify folding intermediates and to investigate interac-
tions with other macromolecules.[2,3,4] Alternatively, com-
plexation of radioactive metals by EDTA,[5,6] as well as
One of the recently applied new tools of structural
chemistry and biology is the use of residual dipolar cou-
plings (RDC) as structural restraints.[8,9,10] Although dipolar
couplings assume values of several kHz in the solid state,
they are normally completely averaged out in isotropic so-
lution. They can be recovered by orienting the molecule ani-
sotropically in solution. In principle, there are two ways to
align proteins; either by putting them into an anisotropic so-
lution, such as dilute liquid crystals,[7] or by changing the
molecule such that it aligns by itself in the external magnetic
field.[8] Anisotropic media can be made from a variety of
sources, as recently reviewed.[11] However, these alignment
media are sometimes not compatible with the compound
under investigation, or they may even bind to the molecule
of interest, thereby precluding its study. Consequently, align-
ment by attaching a paramagnetic tag to the molecule be-
comes attractive. The tag must host a paramagnetic species
that has a fast-relaxing electron spin, is highly anisotropic,
does not interfere with the protein, and has a short linker to
minimize averaging of the anisotropy of the alignment tensor.
Lanthanides have been used successfully in aqueous solu-
tions for the alignment of proteins that have a metal binding
site.[12,13] The clear limitation of this concept is that it can be
applied only to metal-binding proteins; however, due to the
[a] Dr. A. Leonov, B. Voigt, F. Rodriguez-CastaÇeda,
Prof. Dr. C. Griesinger
Max-Planck Institute for Biophysical Chemistry
Department of NMR-Based Structural Biology
Am Fassberg 11, 37077 Gçttingen (Germany)
Fax : (+49)551-201-2200
E-mail: cigr@nmr.mpibpc.mpg.de
[b] Dr. P. Sakhaii
Present address: Aventis Pharma Deutschland GmbH
Process Development Chemistry, PDS Analytics, Building G 838
65926 Frankfurt am Main (Germany)
3342
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DOI: 10.1002/chem.200400907
Chem. Eur. J. 2005, 11, 3342 – 3348

FULL PAPER
rigidity of the binding site with respect to the remainder of
the protein, strong alignment with large dipolar couplings
could be achieved, and coupling constants of up to 40 Hz
were observed.[13] Lanthanide binding sites have been intro-
duced into proteins by attaching either a calmodulin half-
domain[14] or the EDTA compound 1 (Figure 1).[15,16] A lan-
different alignment tensors when attached to a protein. At-
tachment of compound 1 (Figure 1) to a cysteine mutant of
the protein trigger factor[22] yields a doubling of the protein
resonances upon addition of dysprosium metal ions in a JHN
-
coupled 1H,15N-heteronuclear single quantum coherence
(HSQC) spectrum, which is due to the epimeric lanthanide
complexes, as described in detail in reference [18]. This
problem has been demonstrated especially clearly for the
complex of compound 3 with a lanthanide metal,[18] for
which the chiral nitrogen could be identified as the source
of the chirality of the lanthanide complex.
Results and Discussion
Compound 2 was synthesized in five steps (Scheme 1). The
acid 6 was prepared according to a modification of the
method of Arya et al.,[23] both in the racemic as well as in
the enantiomerically pure way. In the first step, commercial-
ly available racemic 2,3-diaminopropionic acid hydrochlo-
ride 4 was exhaustively alkylated with tert-butyl bromoace-
tate in the presence of N-ethyldiisopropylamine to provide
the pentaester 5. The literature conditions[23] for preparing
compound 6 were the incubation of 5 at 1008C in dimethyl-
formamide in the presence of a 1m equivalent of sodium thi-
ophenoxide. Under these reaction conditions, we obtained
only traces of compound 6 in the mixture, and this was very
difficult to purify. Therefore, we changed the deprotecting
agent. The pentaester 5 was selectively saponified without
purification by using lithium hydroxide to provide the car-
boxylic acid 6 in 38% yield for the two steps.
Figure 1. Structures of the EDTA-based metal chelates.
thanide complex, named CLaNP (caged lanthanide NMR
probe), has been developed recently for the characterization
of proteins by paramagnetic NMR spectroscopy;[17] however,
it yields up to five sets of signals due to uncontrolled stereo-
chemistry. The feasibility of the tag proposed in the present
study, which permits alignment yielding only one set of sig-
nals, has been recently shown.[18]
The preparation of the second building block
8
(Scheme 1) has been described by Block and Weidner.[24]
However, the synthetic route utilizes the commercially un-
available reactant methanethiosulfonic acid S-trifluorometh-
yl ester. This ester can be synthesized using the toxic and
commercially unavailable reactant trifluoromethanesulfenyl
chloride. To avoid usage of toxic compounds, a reliable pro-
cedure on a gram-scale for the synthesis of (R)-S-mesylcys-
teine 8 was found by applying the Hart protocol.[25] Treat-
ment of (R)-cysteine hydrochloride hydrate 7 in water with
sodium nitrite and hydrochloric acid afforded the unstable
S-nitrosocysteine in situ. The crude reaction mixture was
treated with sodium methanesulfinate and the target com-
pound 8 was isolated in 55% yield by simple filtration. The
racemic acid 6 was then coupled to (R)-S-mesylcysteine 8 in
dimethylformamide (DMF) by using standard HOBt/DCC
(N-hydroxybenzotriazole/N,N’-dicyclohexylcarbodiimide)
techniques,[26] followed by deprotection of the diastereomer-
ic mixture 9 by trifluoroacetic acid without purification. The
mixture of diastereomeric compounds 2 was subjected to
chromatographic separation (HPLC) to afford pure diaster-
eomers as two peaks with retention times of 9.87 min for
peak 1 and 10.53 min for peak 2, in 30% yield.
The present work describes the synthesis and binding af-
finities of a novel, stereochemically unique EDTA-based tag
that can be attached to a broad range of different proteins
containing one accessible cysteine residue. If no cysteine res-
idue is present in the native protein, it can be introduced by
performing site-directed mutagenesis. The proposed tag has
a high affinity to lanthanides. In addition, it reacts readily
with cysteine side chains in proteins, because of the mesyl
leaving group of the methanethiosulfonyl (MTS) moiety,
this approach has been used before to attach fluorescent
dyes or spin labels to proteins for electron paramagnetic res-
onance (EPR) or relaxation studies. We used EDTA to
retain the high affinity to lanthanides,[19,20,21] and we de-
signed the tag so that no chirality is introduced upon com-
plexation of the tag by the lanthanide. In addition, the
linker between the cysteine residue and the tag should be as
short as possible to minimize conformational averaging and,
therefore, to maximize dipolar couplings.
The most difficult step is obtaining tags that do not
assume different stereoisomers upon complexation of the
lanthanide.[18] This was overcome by attaching the linker
with defined stereochemistry to the C-1 position of the
EDTA, as shown for compounds 2 (Figure 1). Indeed, com-
pound 1 does not have this property and, therefore, shows
The absolute configurations of the 2,3-diaminopropionic
fragments of the two diastereomeric compounds 2 were de-
termined as follows (Scheme 2). The optically active (R)-
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C. Griesinger et al.
Scheme 1. Synthesis of the EDTA-based metal chelates.
Scheme 2. Synthesis of (R,R)-2 for the stereochemical determination of peaks 1 and 2.
2,3-bis[di(tert-butyloxycarbonylmethyl)amino]propionic acid
(R)-6 was synthesized from commercially available (R)-2,3-
diaminopropionic acid hydrochloride by the procedure used
for the synthesis of racemic acid 6. The resulting product
was then coupled to (R)-S-mesylcysteine 8 by using HATU
(N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-
methylene]-N-methylmethanaminium hexafluorophosphate
N-oxide) as the condensation reagent, to give the diaster-
eomer (R,R)-2 in the same yield after deprotection with
formic acid followed by HPLC purification. This compound
with known configuration of both stereogenic centers was
used as a standard for the HPLC analysis of the two diaster-
eomers (R,R)-2 and (S,R)-2 synthesized from racemic acid 6.
By comparing the HPLC traces we can conclude that peak 1
has the same retention time as (R,R)-2. The 1H,13C NMR
spectroscopy and MS data of peak 1 were in agreement with
data from (R,R)-2. Thus, peak 2 is for (S,R)-2.
NMR spectrum. The alignment tensors were rotated relative
to each other, thus inducing linearly independent dipolar
couplings, as well as pseudocontact shifts.
The novel tag molecules, (R,R)-2 and (S,R)-2, provide
only one set of signals after complexation with lanthanide.
This is because strong binding of the lanthanide does not
induce additional chirality in the molecule, due to the nitro-
gen becoming tetrahedral. The two nitrogen atoms have two
identical ligands, namely the carboxymethyl groups. Even
when the nitrogen becomes locked in the tetrahedral config-
uration upon binding of the metal, it does not become
chiral, because the two carboxymethyl groups remain as two
identical ligands. This is the key difference to compound 1
(Figure 1), which becomes chiral at the nitrogen to which
the linker is attached, because there are four different li-
gands: the carboxymethyl, the aminocarbonyl with the
sulfur linker attached, the lanthanide, and the 2-[di(carboxy-
methyl)amino]ethyl. The two chiral conformations of 1 are
shown in Figure 2.
Both the (R,R)-2 (epi-3 in reference [18]) and the (S,R)-2
(3 in reference[18]) were used for the tagging reaction of
the cysteine-mutant trigger factor protein and, due to the
stereochemical purity, yielded only one set of signals in the
The feasibility of the tag for various biomolecular systems
could be established by using ubiquitin and calmodulin. An
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EDTA-Based Chiral Metal Chelates
FULL PAPER
Figure 2. The two chiral conformations of compound 1 upon complexation of the EDTA part with a lanthanide. If the residue R has, for example, the
chirality R, two diastereomeric compounds are formed upon complexation of the lanthanide.
Figure 4. Correlation plots of the observed residual dipolar couplings
(RDC) versus the calculated RDC values from the NMR data of human
ubiquitin (PDB 1D3Z); A) ubiquitin T12C–R,R-2–Tb3+ and B) ubiquitin
T12C–S,R-2–Tb3+
.
tags. A total of 38 and 24 residual dipolar couplings ranging
from À6 to 8 Hz were measured, and a correlation plot of
the measured versus expected dipolar couplings for this
mutant is shown in Figure 4. The pseudocontact shifts as-
sumed values ranging from À0.93 to 0.549 ppm. The two
tensors have an angle in five dimensional space of 658,
which is similar to the angle measured in the case of trigger
factor.[18]
The behavior of tag 2 is different to that of the commer-
cially available compound 1. The latter showed doubling of
the resonances upon complexation with the lanthanide[18]
due to formation of the new chiral center, as shown in
Figure 2. Together with the chiral protein, two diastereomer-
ic compounds are formed that show different physicochemi-
cal properties and, therefore, also different alignment ten-
sors.
To check the versatility of the tag, its binding constant to
lanthanide ions was determined by measuring luminescence
based on the energy transfer of the laser dye cs124 (7-
amino-4-methylquinolin-2(1H)-one)[27] to a proximal lantha-
nide. For this purpose, the racemic acid 6 was attached to
Figure 3. Superimposition of the properly combined IPAP spectra of the
T12C mutant of ubiquitin tagged with (S,R)-2 and complexed with Tb3+
.
The coupling constants and the pseudocontact shifts range between À6
and 8 Hz, and À0.93 and 0.549 ppm, respectively.
in-phase/anti-phase (IPAP) HSQC spectrum of the ubiquitin
T12C mutant attached to (S,R)-2 (3 in reference[18]) com-
plexed with Tb3+ is shown in Figure 3. The doublets in w1
for the tagged species are clean peaks, so that there is only
one alignment tensor induced for this molecule through the
paramagnetic tagging by either of the two diastereomeric
Chem. Eur. J. 2005, 11, 3342 – 3348
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C. Griesinger et al.
Scheme 3. Synthesis of complexes 11 for the determination of stability constants.
tific (BIBBY, UK) capillary apparatus and are uncorrected. Thin layer
chromatography (TLC): Macherey–Nagel precoated sheets, 0.25 mm
Polygram SIL G/UV254 plates, detection with UV and/or by charring with
10 wt% ethanolic phosphomolybdic acid reagent followed by heating at
2008C. Flash column chromatography was performed by using Merck
silica gel 60 (0.015–0.040 mm). Analytical and preparative high perform-
ance liquid chromatography (HPLC) were performed by using a Waters
HPLC system with a Waters 996 Photodiode Array Detector. All separa-
tions involved a mobile phase of 0.1% trifluoroacetic acid (TFA) (v/v) in
water (solvent A) and 0.1% TFA in acetonitrile (solvent B). HPLC was
performed by using a reversed-phase (RP) column Eurospher RP 18,
100 ꢃ, 5 mm, 250ꢀ4.6 mm (analytical) and 250ꢀ16 mm (preparative) at
flow rates of 1 mLminÀ1 (analytical) and 7 mLminÀ1 (preparative). Elec-
trospray ionization mass spectrometry (ESI-MS) and liquid chromatogra-
the laser dye cs124 by using HATU as coupling reagent to
give amide 10. The subsequent deprotection under acidic
conditions and HPLC purification afforded 11 in 83% total
yield (Scheme 3).
The luminescence was measured by using a 1:1 stoichiom-
etry of lanthanide ion and tag 11. The binding constant Kb
for the tag was determined relative to the binding affinity of
EDTA to terbium (Kb =2ꢀ1017 mÀ1) by titrating the complex
with increasing amounts of EDTA according to Oserꢂs pro-
tocol.[28] A 14 mm solution of the 11–Tb3+ complex gave a
fluorescence value of 68 units at the highest emission line at
546 nm in the buffer used for the NMR spectroscopy meas-
urements (50 mm MOPS (3-(N-morpholino)propanesulfonic
acid), 50 mm NaCl, pH 6.8). Upon increasing the EDTA
concentrations, a linear reduction in the luminescence was
observed. The Kb value obtained from the 11–Tb3+ complex
was 6.5ꢀ1017 mÀ1, confirming that the binding of terbium
and other similar lanthanide ions is of the same order of
magnitude as the binding to EDTA. We suggest that the
binding affinity of tag 11 to the Tb3+ ion is three times
higher than the affinity to EDTA, because of the stabilizing
effect of the additional amide group. In addition, no signifi-
cant reduction in the luminescence intensity was seen upon
addition of a 104 excess of Ca2+ ions, indicating that this par-
amagnetic tag is compatible with buffers that contain high
concentrations of divalent alkaline earth ions.
phy/mass spectrometry (LC/MS) analyses were obtained by using
a
Waters Micromass ZQ 4000 mass spectrometer in conjunction with the
Waters HPLC apparatus described above. High-resolution mass spectra
(HRMS) were recorded by using an MS Finnigan LCQ (Ion-Trapp) mass
spectrometer and are reported in m/z. Optical rotations were recorded
by using a Perkin–Elmer 241 digital polarimeter 1 dm cell and are given
in units of 10À1 degcm3 gÀ1 dmÀ1.The terbium luminescence measurements
were obtained by using a Varian Cary Eclipse spectrofluorimeter. Ele-
mental analyses were performed at the Microanalytical Laboratory of the
Institute of Organic Chemistry, Gçttingen, Germany. NMR spectra were
recorded at a temperature of 298 K by using a 400 MHz Bruker Avance
spectrometer (Bruker AG, Rheinstetten, Germany) equipped with a TXI
HCN z-gradient probes header. The IPAP 1H,15N NMR spectra were re-
corded at a temperature of 303 K by using a 900 MHz Bruker Avance
spectrometer. The lyophilized 15N-tagged ((R,R)-2 or (S,R)-2) ubiquitin
was dissolved in the appropriate buffer (50 mm MOPS buffer, 50 mm
NaCl, pH 6.8) with 10% (v/v) D2O for the lock signal. For NMR spectro-
scopy measurements, the 280 ml sample was introduced into a Shigemi
NMR spectroscopy sample tube of 5 mm diameter with an adaptor-
1
piston inserted upward (BMS-3). The H,15N NMR spectra were obtained
with spectral widths of 15 ppm (1H) and 30 ppm (15N). The carrier posi-
Conclusion
1
tions were 115 ppm for 15N and 4.7 ppm for H. The IPAP-HSQC experi-
ments were acquired with 2048 and 512 complex points in t2 and t1, re-
spectively. The processing was performed by using NMR Pipe.[29] Zero
filling in the indirect dimension to 8192 points led to a digital resolution
of 0.33 Hz/point. All other spectra were processed by using
XWINNMR 3.1 (Bruker AG, Karlsruhe, Germany). 1H NMR chemical
shifts (d) are reported in parts per million (ppm) relative to tetramethyl-
silane (TMS) (d=0.00 in CDCl3), [D5]DMSO (d=2.49 in [D6]DMSO),
and 4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt (DSS) (d=
0.00 in D2O) as internal standards. Data are reported as follows: chemi-
cal shift, multiplicity (s=singlet, d=doublet, t=triplet, br=broadened,
m=multiplet), coupling constants (J, given in Hz), integration. 13C NMR
chemical shifts (d) are reported in parts per million (ppm) relative to
CDCl3 (d=77.0), [D6]DMSO (d=39.7), and DSS (d=0.00 in D2O) as in-
ternal standards. The following two dimensional (2D) experiments were
used for resonance assignments: 2D-[13C,1H]-HSQC (heteronuclear single
quantum coherence),[30] 2D-[13C,1H]-HMBC (heteronuclear multiple
bond correlation).[31]
We have described an efficient synthesis of two diastereo-
meric EDTA-based tag molecules that can be attached to
the cysteine residue of a protein. The tags are stereochemi-
cally pure, so that they induce only one set of signals once
loaded with a lanthanide. The two diastereomers induce lin-
early independent alignment tensors, which is advantageous
for the determination of protein structure. The binding af-
finity is six times higher than that for EDTA, which facili-
tates the use of the tag even for metal-binding proteins, such
as calmodulin.
Experimental Section
2,3-Bis[di(tert-butoxycarbonylmethyl)amino] propionic acid (6): tert-
Butyl bromoacetate (6.83 mL, 9.02 g, 46.2 mmol) was added to a suspen-
sion of 2,3-diaminopropionic acid hydrochloride 4 (1.00 g, 7.1 mmol) and
N-ethyldiisopropylamine (DIPEA) (8.52 mL, 6.44 g, 49.8 mmol) in aceto-
General: Starting materials and solvents were commercially available
unless otherwise noted. All reactions were conducted under an atmos-
phere of argon. Melting points were determined by using a Stuart Scien-
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EDTA-Based Chiral Metal Chelates
FULL PAPER
nitrile (50 mL). After stirring at reflux for 16 h, additional tert-butyl bro-
moacetate (1.05 mL, 1.39 g, 7.1 mmol), and DIPEA (1.22 mL, 0.92 g,
7.1 mmol) were added. The reaction mixture was refluxed for 32 h,
cooled, and the solvent was removed under reduced pressure. The resi-
due was mixed with diethyl ether (100 mL), refluxed for 1 h, cooled, and
filtered. The filtrate was washed with 0.1m phosphate buffer pH 2.0 (3ꢀ
40 mL), dried (molecular sieve 0.4 nm powder), and concentrated under
reduced pressure. The residue was dissolved in tetrahydrofuran (60 mL),
and lithium hydroxide (1m solution in water, 7.1 mL, 7.1 mmol) was
added. After stirring at room temperature for 3 h, additional LiOH (1m
solution in water, 3.6 mL, 3.6 mmol) was added, and stirring was contin-
ued for 2 h. Acetic acid (0.63 mL, 0.66 g, 11 mmol) was added and the
solvent was removed under reduced pressure. Phosphate buffer (0.1m,
pH 2.0, 40 mL) was added and the mixture was extracted with chloro-
form (3ꢀ10 mL). The organic phase was dried (molecular sieve 0.4 nm
powder) and concentrated under reduced pressure. The residue was puri-
fied by performing silica gel chromatography (10:1 chloroform/methanol,
Rf =0.4 (streak)) to afford 6 (1.52 g, 2.7 mmol, 38%) as a slightly yellow
oil. 1H NMR and MS data were in agreement with literature data.[22,25]
13C NMR (100.6 MHz, CDCl3): d=28.47 (12ꢀC, 4ꢀ(CH3)3C), 55.04 (2ꢀ
C, CH2CO2tBu), 55.28 (C-3), 57.03 (2ꢀC, CH2CO2tBu), 63.45 (C-2),
82.11 (4ꢀC, (CH3)3C), 170.43 (2ꢀC, CH2CO2tBu), 171.85 (2ꢀC,
CH2CO2tBu), 173.62 ppm (C-1).
under reduced pressure, the residue redissolved in ethyl acetate (10 mL),
washed with 0.1m phosphate buffer pH 2.0 (2ꢀ5 mL), dried (molecular
sieve 0.4 nm powder), and concentrated under reduced pressure. The res-
idue was dissolved in a deprotection mixture (CH2Cl2/TFA, 1:1, v/v,
20 mL). After stirring for 20 h the solvent was removed by evaporation
under reduced pressure. The oily residue was redissolved in acetonitrile/
water (1:1, 30 mL), filtered (0.45 mm nylon filter), and lyophilized to give
a colorless foam. The crude diastereomeric mixture was separated by
RP-HPLC (gradient 0–10% solvent B in 30 min, RT ~10–12 min) to
afford both diasteromers. The combined fractions showing pure material
were lyophilized, affording peak 1 (R,R-2) (38 mg, 74 mmol, 15%; analyti-
cal HPLC 0–10% solvent B in 30 min, retention time (RT) 9.87 min) and
peak 2 (S,R-2) (39 mg, 75 mmol, 15%; analytical HPLC 0–10% solvent B
in 30 min, RT 10.53 min).
N-[(R)-2,3-Bis[di(carboxymethyl)amino]propionyl]-S-mesyl-(R)-cysteine
(peak 1, (R,R)-2): 1H NMR (400 MHz, D2O, HSQC, HMBC): d=3.44 (s,
3H; CH3), 3.56 (dd, J=14.6, 7.0 Hz, 1H; Cys-HA-3), 3.62 (m, 1H; HA-3’),
3.65 (dd, J=14.6, 4.7 Hz, 1H; Cys-HB-3), 3.69 (m, 1H; HB-3’), 3.70 (s,
4H; 2ꢀCH2CO2H), 4.04 (dd, J=10.2, 4.9 Hz, 1H; H-2’), 4.18 (d, J=
17.3 Hz, 2H; CH2CO2H), 4.21 (d, J=17.3 Hz, 2H; CH2CO2H), 4.78 ppm
(dd, J=7.0, 4.7 Hz, 1H; Cys-H-2); 13C NMR (100.6 MHz, D2O): d=35.94
(Cys-C-3), 49.28 (CH3), 51.66 (2ꢀC, CH2CO2H), 51.82 (Cys-C-2), 54.01
(C-3’), 55.62 (2ꢀC, CH2CO2H), 59.29 (C-2’), 168.38 (2ꢀC, CH2CO2H),
169.67 (C-1’), 171.64 (Cys-C-1), 174.99 ppm (2ꢀC, CH2CO2H); ESI-
HRMS (MeOH, positive mode): calcd for C15H24N3O13S2 [M+H]+:
518.07451; found: 518.07434.
(R)-2,3-Bis[di(tert-butoxycarbonylmethyl)amino] propionic acid ((R)-6):
In a similar manner, (R)-2,3-diaminopropionic acid hydrochloride (2.00 g,
14.2 mmol) gave R-6 (3.31 g, 5.9 mmol, 41%) as a white solid. M.p. 66–
698C; [a]2D0 =+15.7 (c=1.00 in methanol); 1H NMR (400 MHz, CDCl3,
HSQC, HMBC): d=1.45 (s, 36H; 4ꢀOtBu), 3.12 (d, J=7.4 Hz, 2H; H-
3), 3.45 (d, J=17.1 Hz, 2H; CH2CO2tBu), 3.50 (d, J=17.6 Hz, 2H;
CH2CO2tBu), 3.56 (d, J=17.1 Hz, 2H; CH2CO2tBu), 3.59 (d, J=17.6 Hz,
2H; CH2CO2tBu), 3.72 (t, J=7.4 Hz, 1H; H-2), 12.20 ppm (brs, 1H;
CO2H); 13C NMR (100.6 MHz, CDCl3): d=28.45 (6ꢀC, 2ꢀ(CH3)3C),
28.50 (6ꢀC, 2ꢀ(CH3)3C), 55.04 (2ꢀC, CH2CO2tBu), 55.28 (C-3), 57.03
(2ꢀC, CH2CO2tBu), 63.45 (C-2), 82.08 (2ꢀC, (CH3)3C), 82.16 (2ꢀC,
(CH3)3C), 170.43 (2ꢀC, CH2CO2tBu), 171.85 (2ꢀC, CH2CO2tBu),
173.62 ppm (C-1); ESI-MS m/z (MeOH, positive mode): calcd for
C27H49N2O10 [M+H]+: 561.68; found: 561.30; elemental analysis calcd
(%) for C27H48N2O10·0.5CHCl3 (620.37): C 53.24, H 7.88; found: C 53.32,
H 8.02.
N-[(S)-2,3-Bis[di(carboxymethyl)amino]propionyl]-S-mesyl-(R)-cysteine
(peak 2, (S,R)-2): 1H NMR (400 MHz, D2O, HSQC, HMBC): d=3.47 (s,
3H; CH3), 3.57 (dd, J=14.7, 7.6 Hz, 1H; Cys-HA-3), 3.64 (dd, J=10.0,
3.8 Hz, 1H; HA-3’), 3.67 (d, J=18.1 Hz, 2H; CH2CO2H), 3.71 (dd, J=
10.0, 5.2 Hz, 1H; HB-3’), 3.72 (d, J=18.1 Hz, 2H; CH2CO2H), 3.73 (dd,
J=14.7, 4.4 Hz, 1H; Cys-HB-3), 4.05 (dd, J=10.0, 5.2 Hz, 1H; H-2’), 4.12
(d, J=17.1 Hz, 2H; CH2CO2H), 4.16 (d, J=17.1 Hz, 2H; CH2CO2H),
4.76 ppm (dd, J=7.6, 4.4 Hz, 1H; Cys-H-2); 13C NMR (100.6 MHz,
D2O): d=36.44 (Cys-C-3), 49.83 (CH3), 52.25 (2ꢀC, CH2CO2H), 52.34
(Cys-C-2), 54.37 (C-3’), 56.28 (2ꢀC, CH2CO2H), 59.73 (C-2’), 169.12 (2ꢀ
C, CH2CO2H), 170.29 (C-1’), 172.41 (Cys-C-1), 175.29 ppm (2ꢀC,
CH2CO2H); ESI-HRMS (MeOH, positive mode): calcd for
C15H24N3O13S2 [M+H]+: 518.07451; found: 518.07468.
(R)-S-Mesylcysteine (8): An ice-cold solution of sodium nitrite (1.38 g,
20.0 mmol) in water (10 mL) was slowly added dropwise at 58C to a stir-
N-[(R)-2,3-Bis[di(carboxymethyl)amino]propionyl]-S-mesyl-(R)-cysteine
((R,R)-2): Compound (R)-6 (280 mg, 500 mmol), HATU (190 mg,
500 mmol), and DIPEA (86 mL, 65 mg, 500 mmol) were preincubated in
DMF (5 mL) and dichloromethane (2 mL). After 10 min, 7 (120 mg,
600 mmol) and DIPEA (120 mL, 90 mg, 700 mmol) dissolved in DMF
(5 mL) were added, and stirring was continued for 21 h. The solvent was
removed under reduced pressure, the residue redissolved in ethyl acetate
(30 mL), washed with 0.1m phosphate buffer pH 2.0 (2ꢀ10 mL), dried
(molecular sieve 0.4 nm powder), and concentrated under reduced pres-
sure. The residue was dissolved in formic acid (10 mL). After stirring for
49 h, toluene (10 mL) was added, and the solvent was removed by evapo-
ration under reduced pressure. The oily residue was redissolved in aceto-
nitrile/water (20:1, 21 mL), filtered (0.45 mm nylon filter), and lyophilized
to give a colorless foam. The crude product was purified by performing
RP-HPLC (gradient 0–30% solvent B in 30 min, RT ~10–12 min) to
afford the pure compound. The combined fractions showing pure materi-
al were lyophilized, affording R,R-2 (76 mg, 147 mmol, 29%) as a white
powder. Analytical HPLC was performed (0–10% solvent B in 30 min,
RT 9.87 min). 1H NMR, 13C NMR and MS data were in agreement with
data for R,R-2 synthesized using racemic acid 6.
red solution of (R)-cysteine hydrochloride monohydrate
7 (3.51 g,
20.0 mmol) in 2n HCl (20 mL). After 1 h, the deep red solution was
treated with sodium methanesulfinate (4.08 g, 40.0 mmol). After stirring
at 58C for 2 h, additional sodium methanesulfinate (1.09 g, 10.0 mmol)
was added and the reaction mixture was stirred for 12 h at 58C. The re-
sulting precipitate was filtered off, then washed with ice-cold water (2ꢀ
5 mL) and diethyl ether (2ꢀ5 mL). Drying in vacuo at 208C afforded
pure (R)-S-mesylcysteine 8 (2.21 g, 11.0 mmol, 55%) as a white solid.
M.p. 146–1498C (decomp); [a]2D0 =À61.7 (c=1.03 in water); 1H NMR
(400 MHz, D2O, HSQC, HMBC): d=3.56 (s, 3H; CH3), 3.71 (dd, J=
15.8, 7.1 Hz, 1H; HA-3), 3.79 (dd, J=15.8, 4.7 Hz, 1H; HB-3), 4.18 ppm
(dd, J=7.1, 4.7 Hz, 1H; H-2); 13C NMR (100.6 MHz, D2O): d=35.81 (C-
3), 50.73 (CH3), 53.44 (C-2), 170.07 ppm (C-1); ESI-HRMS (MeOH, pos-
itive mode): calcd for C4H10NO4S2 [M+H]+: 200.00458; found:
200.00469; elemental analysis calcd (%) for C4H9NO4S2 (199.25): C 24.11,
H 4.55; found: C 24.14, H 4.36.
N-[(R)-2,3-Bis[di(carboxymethyl)amino]propionyl]-S-mesyl-(R)-cysteine
((R,R)-2)
and
N-[(S)-2,3-bis[di(carboxymethyl)amino]propionyl]-S-
mesyl-(R)-cysteine ((S,R)-2): DCC (1m solution in dichloromethane,
500 mL, 500 mmol) was added at 58C to a stirred solution of racemic acid
6 (280 mg, 500 mmol) and HOBt·H2O (77 mg, 500 mmol) in dichlorome-
thane (3 mL) and DMF (1.5 mL). The reaction mixture was stirred at
58C for 1 h and at 208C for 2 h, followed by filtration. The filtrate was
evaporated to dryness, and the residue was redissolved in dichlorome-
2,3-Bis[di(tert-butoxycarbonylmethyl)amino]-N-(4-methyl-2-oxo-1,2-dihy-
droquinolin-7-yl)propanamide (10): Compound 6 (667 mg, 1.19 mmol),
HATU (494 mg, 1.30 mmol), and DIPEA (204 mL, 154 mg, 1.19 mmol)
were preincubated in dimethyl sulfoxide (5 mL). After 10 min, cs124
(174 mg, 1.00 mmol) was added, and stirring was continued for 17 h.
Ethyl acetate (50 mL) was added and the mixture was washed with 0.1m
phosphate buffer pH 2.0 (2ꢀ20 mL), dried (molecular sieve 0.4 nm
powder), and concentrated under reduced pressure. The residue was puri-
fied by performing silica gel chromatography (10:1 chloroform/methanol,
thane (2 mL) and DMF (5 mL). (R)-S-Mesylcysteine
8 (141 mg,
707 mmol) and DIPEA (513 mL, 388 mg, 3.0 mmol) were added and the
reaction mixture was stirred 14 h at 208C. The solvent was removed
Chem. Eur. J. 2005, 11, 3342 – 3348
www.chemeurj.org
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3347

C. Griesinger et al.
Rf =0.64) to afford 10 (680 mg, 0.95 mmol, 95%) as a white solid. M.p.
80–838C. 1H NMR (400 MHz, CDCl3, HSQC, HMBC): d=1.45 (s, 36H;
4ꢀOtBu), 2.44 (s, 3H; CH3-C-4’), 3.00 (dd, J=14.2, 7.7 Hz, 1H; HA-3),
3.38 (dd, J=14.2, 5.6 Hz, 1H; HB-3), 3.43 (d, J=17.6 Hz, 2H; CH2CO2t-
Bu), 3.590 (d, J=17.6 Hz, 2H; CH2CO2tBu), 3.593 (s, 4H; 2ꢀCH2CO2t-
Bu), 3.76 (t, J=7.3 Hz, 1H; H-2), 6.40 (s, 1H; H-3’), 7.39 (d, J=8.6 Hz,
1H; H-6’), 7.57 (d, J=8.6 Hz, 1H; H-5’), 7.85 (s, 1H; H-8’), 9,50 (brs,
1H; HN-C-2’), 10.92 ppm (brs, 1H; HN-C-1); 13C NMR (100.6 MHz,
CDCl3): d=19.01 (CH3-C-4’), 28.12 (12ꢀC, 4ꢀ(CH3)3C), 54.25 (C-3),
54.43 (2ꢀC, CH2CO2tBu), 56.53 (2ꢀC, CH2CO2tBu), 65.10 (C-2), 81.15
(2ꢀC, (CH3)3C), 81.47 (2ꢀC, (CH3)3C), 104.83 (C-8’), 114.54 (C-6’),
116.52 (C-4a’), 119.08 (C-3’), 125.15 (C-5’), 138.80 (C-8a’), 140.86 (C-7’),
148.42 (C-4’), 163.03 (C=O, C-2’), 170.65 (2ꢀC, CO2tBu), 171.74 (2ꢀC,
CO2tBu), 171.99 ppm (C=O, C-1); ESI-MS (MeOH, positive mode):
calcd for C37H57N4O10 [M+H]+: 717.8; found: 717.4; elemental analysis
calcd (%) for C37H56N4O10·0.1CHCl3 (728.82): C 61.14, H 7.76, N 7.69;
found: C 61.01, H 7.51, N 7.30.
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2,3-Bis[di(carboxymethyl)amino]-N-(4-methyl-2-oxo-1,2-dihydroquinolin-
7-yl)propanamide (11): Compound 10 (773 mg, 1.07 mmol) was dissolved
in a deprotection mixture (TFA/CH2Cl2/iPr3SiH, 5:5:1.8, v/v, 11.8 mL).
After stirring for 16 h, toluene (10 mL) was added, and the solvent was
removed by evaporation under reduced pressure. Ethyl acetate (5 mL)
was added and the heterogeneous mixture was stirred for 1 h at 408C.
The resulting precipitate was filtered off, washed with ethyl acetate (2ꢀ
5 mL), and mixed with water (20 mL). Lyophilization afforded the pure
compound 11 (492 mg, 0.93 mmol, 87%) as a white solid. M.p. 2408C
(decomp). 1H NMR (400 MHz, CDCl3, HSQC, HMBC): d=2.36 (s, 3H;
CH3-C-4’), 3.09 (dd, J=13.6, 8.3 Hz, 1H; HA-3), 3.22 (dd, J=13.6, 6.0 Hz,
1H; HB-3), 3.56 (d, J=17.9 Hz, 2H; CH2CO2H), 3.625 (s, 4H; 2ꢀ
CH2CO2H), 3.631 (d, J=17.9 Hz, 2H; CH2CO2H), 3.78 (t, J=7.2 Hz,
1H; H-2), 6.25 (s, 1H; H-3’), 7.24 (d, J=8.7 Hz, 1H; H-6’), 7.62 (d, J=
8.7 Hz, 1H; H-5’), 7.82 (s, 1H; H-8’), 10,65 (brs, 1H; HN-C-1), 11.53
(brs, 1H; HN-C-2’), 12.40 ppm (brs, 4H; CO2H); 13C NMR (100.6 MHz,
CDCl3): d=18.84 (CH3-C-4’), 53.58 (2ꢀC, CH2CO2H), 54.35 (C-3), 54.85
(2ꢀC, CH2CO2H), 63.36 (C-2), 104.96 (C-8’), 114.06 (C-6’), 116.06 (C-
4a’), 119.48 (C-3’), 125.69 (C-5’), 139.94 (C-8a’), 140.82 (C-7’), 148.02 (C-
4’), 162.43 (2ꢀC, CO2H), 170.96 (C=O, C-1), 172.26 (C=O, C-2’),
173.95 ppm (2ꢀC, CO2H); ESI-MS (MeOH, positive mode): calcd for
C21H25N4O10 [M+H]+: 493.4; found: 493.0; elemental analysis calcd (%)
for C21H24N4O10·2H2O (528.47): C 47.73, H 5.34; found: C 47.88, H 5.18.
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Acknowledgements
This work was supported by the MPG, the DFG (SFB 492 Z-project) and
the Fonds of the Chemical Industry. We thank Mrs Kerstin Overkamp
for performing the HPLC purification and for ESI-MS analyses. We ac-
knowledge measurement of ESI-HRMS spectra by Dr. Holm Frauendorf,
University of Gçttingen. Frank Hambloch, University of Gçttingen, is ac-
knowledged for conducting the elemental analyses and Olaf Senge, Uni-
versity of Gçttingen, for chiral HPLC and GC analyses. We acknowledge
Dr. Rolf Vermeij for fruitful discussions regarding the terbium lumines-
cence measurements. Finally, we would like to thank Dr. Laurent Ver-
dier, University of Reims, who was involved in the early stages of this
work.
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Received: September 3, 2004
Published online: March 30, 2005
3348
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 3342 – 3348