Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

Article abstract of DOI:10.1002/cphc.201900211

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for ¹H and ¹³C (¹H→¹³C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for ¹H and 13.9±0.5 for ¹³C (direct polarization).

Full text of DOI:10.1002/cphc.201900211

Accepted Article
Title: Efficient building-blocks for solid state peptide synthesis of spin-
labeled peptides for EPR and DNP applications
Authors: Martin Brodrecht, Kevin Herr, Sarah Bothe, Marcos de
Oliveira Jr., Torsten Gutmann, and Gerd Buntkowsky
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To be cited as: ChemPhysChem 10.1002/cphc.201900211
Link to VoR: http://dx.doi.org/10.1002/cphc.201900211
A Journal of
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ChemPhysChem
10.1002/cphc.201900211
Efficient building-blocks for solid state peptide
synthesis of spin-labeled peptides for EPR and
DNP applications
a
a
a
a
Martin Brodrecht , Kevin Herr , Sarah Bothe , Dr. Marcos de Oliveira Jr. , PD Dr.
a,b
a
Torsten Gutmann and Prof. Dr. Gerd Buntkowsky *
a
Institut für Physikalische Chemie, Technische Universität Darmstadt, 64287 Darmstadt,
Germany
b
University Kassel, Institute for Chemistry, Heinrich-Plett-Straße 40, D-34132 Kassel
*eMail: gerd.buntkowsky@chemie.tu-darmstadt.de
Abstract:
Specific spin labeling allows the site selective investigation of biomolecules by EPR and DNP enhanced
NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is
developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain
of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step
synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the
Fmoc-protection group, which makes them applicable for the use in solid phase peptide synthesis
(SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which
makes it more versatile than the widely used post synthetic spin labeling strategies. For the final
building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments
1
employing these building-blocks in a TCE solution show enhancement factors of up to 26 for H and
1
3
1
13
C ( H C cross-polarization). To proof the viability of the presented building-blocks for insertion of
the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly peptide was
synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated
and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors
1
13
of 12.1±0.1 for H and 13.9±0.5 for C (direct polarization).
Keywords:
spin labeling; solid-state NMR; SPPS, amino acids; EPR; DNP NMR; hyperpolarization
1
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ChemPhysChem
10.1002/cphc.201900211
1
Introduction
Structure determination of biological molecules, such as peptides or proteins is a major challenge in
biological, chemical and medical research. In the last years, methods like high-resolution dynamic
nuclear polarization (DNP) solid state nuclear magnetic resonance (ssNMR)1-6 and electron
paramagnetic resonance (EPR)7-11 significantly contributed to the structural characterization of these
biomolecules. Both techniques require a paramagnetic residue within the sample and in order to perform
local analyses, these paramagnetic residues must also have a specific location in the molecule.12, 13
The most frequently used organic spin labels consist of nitroxyl derived radicals such as (2,2,6,6-
Tetramethylpiperidin-1-yl)oxyl (TEMPO) or (2,2,5,5-Tetramethylpyrrolidin-1-yl)oxyl (PROXYL),
respectively their derivates. The site specific insertion of these paramagnetic centers into a biomolecule,
the so-called spin labeling, is a major challenge in organic chemistry and biochemistry. The most
common way to spin label peptides or proteins is a post synthetic approach. In these approach, the target
molecules are synthesized chemically or biologically and the spin label is chemically attached after the
synthesis. While this approach seems conceptually easy, it is in reality challenging due to the variety of
functional groups exposed in the large amount of amino acids present in these biomolecules. In order to
achieve a specific labeling at one position, a unique binding site, e.g. a unique amino acid, must be
inserted at the targeted position in the peptide or protein. Even when this challenging step is achieved,
the post synthetic coupling of the spin label often suffers from low conversion or poor selectivity.14, 15
For peptides, proteins and enzymes mostly cysteine sites are used for subsequent functionalization.16-22
An alternative post-synthetic approach is the labelling via tyrosine residues.23, 24
2
5
A conceptually elegant approach to solve the labeling problem was proposed by Vitzhum et al. They
first synthesize the desired peptide without spin-label by solid phase peptide synthesis and afterwards
add the spin label to the C-terminus at the free carboxyl-group of the cleaved peptide. The drawback of
this approach is that the spin label can be attached only at one end (C-terminus) of the peptide chain and
not in any position inside the chain, which renders it less attractive e.g. for EPR applications like
PELDOR/DEER.10, 26-30
Another post-synthetic approach employs "click-chemistry" based synthesis strategies, due to their
selectivity to certain functional groups, which are not present in any natural amino acid.31, 32 However
click-chemistry requires special functional precursors for the coupling including reactive groups such
as azides (N=N=N) or alkynes (C≡C). These precursors have to be inserted in the form of a non-natural
amino acid during peptide/protein synthesis, which is mostly done by solid phase peptide synthesis
(SPPS). Afterwards the specific functional group is coupled to the corresponding functional group at the
spin label. These precursors are often expensive or not commercially available at all. Moreover, although
3
3
34
there are copper free approaches , which were successfully employed for orthogonal spin-labeling
common click-chemistry uses a copper catalyst, which is not compatible with many biological
systems.32, 35
An alternative approach to post-synthetic modification is the insertion of the spin label during synthesis
of the target molecule, employing e.g. non-natural amino acid radicals as spin label. Until now, this
approach is not widely used, since the spin label must preserve its paramagnetic character through the
whole synthesis and purification process of SPSS. The only reported radical spin label, which fulfills
this criterion is Fmoc-TOAC 01 (Figure 1).36-40 Since the strong geometric constraints of the TOAC-
radical influence the secondary structure of the peptide backbone, its application is restricted to those
cases, 7, 14, 41 where the secondary structure of the peptide/protein close to the spin label is of minor
concern to the spectroscopic question.
2
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ChemPhysChem
10.1002/cphc.201900211
Figure 1: Chemical structure of the commonly used TOAC (left) and the schematic structure of a side chain functionalized
amino acid (right).
For these reasons, we decided to develop a robust and versatile spin-labeling strategy, where the peptide
conformation and configuration is only minimally distorted. Our approach is based on non-natural
amino acid radicals, which carry a stable radical on its side chain 02 and can be used in SPPS (Figure
1
). In this work, an efficient way to modify hydroxyl side chains of amino acids (hydroxyproline, serine,
tyrosine) with a PROXYL radical is presented, which makes them applicable in SPPS as a building-
block. This approach allows the introduction of the spin label at any position in a peptide or protein
without weakening the secondary structure seriously. Furthermore, it eases the synthesis of peptide-
based bio-compatible radicals for DNP applications by SPPS. As a requirement, the SPPS building-
block needs to carry a Fmoc-protection group at its nitrogen and must have an accessible COOH group
to couple it during the SPPS. The general synthetic pathway to achieve the PROXYL radical coupling
to the hydroxyl group of an amino acid by preserving the Fmoc-group at the N-terminus is illustrated in
Scheme 1. The first reaction step is to protect the carboxylic group of the amino acid 03 to avoid
intermolecular reactions during the next reaction steps. The protected amino acid 04 is coupled then in
an esterification reaction with 3-carboxy-PROXYL 05 to obtain the spin labeled amino acid 06. After
t
that, the Bu protection group has to be cleaved to recover the carboxylic group resulting in the final
amino acid building-block 07. Finally, the obtained building-block 07 could be incorporated into any
peptide of choice at any position by SPPS resulting in the spin labeled peptide 08.
Scheme 1: General synthesis of the PROXYL functionalized amino acid building-block 07 by protection of the amino acid
t
0
3, esterification reaction with 3-carboxy-PROXYL 05 and final deprotection of the Bu ester 06. Standard SPPS protocols
can be used to obtain the spin labeled peptide 08.
The success of our strategy is demonstrated for the commercially available Fmoc-amino acids
hydroxyproline, serine, tyrosine that are modified with a stable radical via their OH side chain. EPR and
DNP NMR are performed to show that the radicals at the building-block side chains stay active after all
3
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ChemPhysChem
10.1002/cphc.201900211
reaction steps. It is also demonstrated that the presented building-blocks are feasible for the use in SPPS.
Therefore, one building-block is incorporated into a small peptide by a standard SPPS protocol and the
spin label activity in the peptide is proved by EPR and DNP NMR measurements.
2
Results and discussion
2.1
Synthesis of the PROXYL side chain modified amino acids
t
t
t
The synthesis of the Bu protected amino acids Fmoc-NH-Hyp(OH)-O Bu 10, Fmoc-NH-Ser(OH)-O Bu
1
(
t
1, Fmoc-NH-Tyr(OH)-O Bu 12 was adapted from the synthesis strategy described in the literature
Scheme 2).42, 43 Tert-butyl 2,2,2-trichloroacetimidate 09 was used to protect the carboxylic group of the
appropriate amino acids Fmoc-NH-Hyp(OH)-OH 13, Fmoc-NH-Ser(OH)-OH 14, Fmoc-NH-Tyr(OH)-
OH 15. To ensure full conversion of the carboxylic group, a large excess of acetimidate 09 and long
reaction times were chosen. These conditions allowed us to use the crude product without further
purification in the next reaction step. This conversion product, however, does not interfere with the next
reaction step.
t
t
t
Scheme 2: Synthesis of Bu protected amino acids Fmoc-NH-Hyp(OH)-O Bu 10, Fmoc-NH-Ser(OH)-O Bu 11, Fmoc-NH-
t
Tyr(OH)-O Bu 12 by the reaction of the Fmoc-amino acids Fmoc-NH-Hyp(OH)-OH 13, Fmoc-NH-Ser(OH)-OH 14, Fmoc-
NH-Tyr(OH)-OH 15 with acetimidate 09.
t
The synthesis of the spin-labeled amino acids Fmoc-NH-Hyp(OOC-Proxyl)-O Bu 16, Fmoc-NH-
t
t
Ser(OOC-Proxyl)-O Bu 17, Fmoc-NH-Tyr(OOC-Proxyl)-O Bu 18 was achieved by an adapted
4
4
synthesis strategy from the literature (Scheme 3) The esterification between the amino acids 10, 11,
2 hydroxyl groups and the 3-carboxy-PROXYL 05 spin label was realized by using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimid hydrochloride 19 (EDC×HCl) and 4-dimethylaminopyridine 20
DMAP) as coupling agents. The reaction for all three amino acids were successfully performed with
1
(
minimal adjustments of the reaction conditions. Excellent yields ranging from 65% to 91% were found.
Both coupling agents could be removed after the reaction by aqueous extraction. Minimal impurities of
the urea derivative from converted EDC do not interfere in the next reaction step.
4
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ChemPhysChem
10.1002/cphc.201900211
t
Scheme 3: Synthesis of the side chain PROXYLated amino acids Fmoc-NH-Hyp(OOC-Proxyl)-O Bu 16, Fmoc-NH-
t
t
t
Ser(OOC-Proxyl)-O Bu 17, Fmoc-NH-Tyr(OOC-Proxyl)-O Bu 18 by the esterification reaction of the Bu protected amino
t
t
t
acids Fmoc-NH-Hyp(OH)-O Bu 10, Fmoc-NH-Ser(OH)-O Bu 11, Fmoc-NH-Tyr(OH)-O Bu 12 with 3-Carboxy-PROXYL
5.
0
The final building-blocks Fmoc-NH-Hyp(OOC-Proxyl)-OH 21, Fmoc-NH-Ser(OOC-Proxyl)-OH 22
t
and Fmoc-NH-Tyr(OOC-Proxyl)-OH 23 have been obtained by Bu deprotection of the esters 16, 17
4
5
and 18 in a DCM/TFA mixture according to the literature. The reaction was successful for all amino
acids in good yields. Although, after this final step, a precise column chromatographic purification had
to be performed for all products since their usage in SPPS and spin labeling applications requires high
purities. This slightly reduces the yield but results in purities obtained by HPLC (ESI) of >99% for all
final building-blocks.
5
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ChemPhysChem
10.1002/cphc.201900211
Scheme 4: Synthesis of the amino acid building-blocks Fmoc-NH-Hyp(OOC-Proxyl)-OH 21, Fmoc-NH-Ser(OOC-Proxyl)-
t
OH 22, Fmoc-NH-Tyr(OOC-Proxyl)-OH 23 by deprotection of the carboxylic group of the Bu esters Fmoc-NH-Hyp(OOC-
t
t
t
Proxyl)-O Bu 16, Fmoc-NH-Ser(OOC-Proxyl)-O Bu 17, Fmoc-NH-Tyr(OOC-Proxyl)-O Bu 18.
2.2
Proof of radical activity by EPR and solid-state DNP NMR
Despite the paramagnetic character of the spin label, solution-state NMR characterization after the
esterification reaction between the amino acids 10, 11, 12 and the spin label 3-carboxy-PROXYL 05
1
gives first hints, whether the spin label was correctly coupled to the amino acid. Therefore, H-NMR
measurements of the PROXYLated species 16, 17, 18 were performed and compared with the spectra
before the spin label was attached (Figure 2). By comparing the spectra of all amino acids before (black)
and after (blue) introduction of the spin-label a broadening of all signals is obtained. This broadening is
4
6-
related to the paramagnetic character of the spin label and clearly indicates that the spin label is active.
5
0
Furthermore, the comparison shows that all signals are preserved during the coupling step. Some
signals are shifted which implies a proximity of these nuclei to the radical in the sample.48, 51, 52 This
particularly applies to hydrogens close to the spin label such as the α (β, γ δ) groups of the amino acids.
The signals of the Fmoc-group are also broadened but not shifted because they are weaker influenced
by the spin label. Since the signal broadening comes along with a decrease in resolution the assignment
and therefore the structural analysis of the spin labeled amino acids becomes challenging. Therefore,
the proof of covalent binding of the spin label is analyzed in detail by mass spectrometry, since the
amino acids can be reliably analyzed by electrospray ionization mass spectrometry (ESI-MS) under mild
ionization without fragmentation. For all synthesized building-blocks the expected masses were found
+
+
in the ESI-MS spectra ([M+H] = 577.3 m/z for Hyp, [M+H] = 551.3 m/z for Ser and
+
[
M+H] = 551.3 m/z for Tyr), which exactly fit the calculated masses including their corresponding
isotope pattern (ESI S20, S22 and S24). In addition, also doubly protonated [M+2H] and sodium
+
+
adducts [M+Na] were found.
6
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ChemPhysChem
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1
Figure 2: Comparison of the H-NMR spectra before and after the PROXYL coupling for each amino acid recorded at RT
t
t
and 500 MHz. a) Fmoc-NH-Hyp(OH)-O Bu 10 (black), Fmoc-NH-Hyp(OOC-PROXYL)-O Bu 16 (blue); b) Fmoc-NH-
t
t
t
Ser(OH)-O Bu 11 (black), Fmoc-NH-Ser(OOC-PROXYL)-O Bu 17 (blue); c) Fmoc-NH-Tyr(OH)-O Bu 12 (black), Fmoc-
NH-Tyr(OOC-PROXYL)-O Bu 18 (blue).
t
After the final deprotection step, the purified building-blocks were analyzed by EPR to probe their
paramagnetic activity. Figure 3 shows EPR spectra for all three paramagnetic building-blocks (21, 22,
2
3) and for the 3-carboxyl-PROXYL precursor (05), measured at 298 K (black curves in Figure 3).
Samples were diluted in 1,1,2,2-Tetrachlorethan (TCE) at concentrations of 1 mM. All substrate amino
acids (13, 14, 15) and protected amino acids before PROXYL coupling (10, 11, 12) do not show any
EPR signal. The EPR spectra for all PROXYL-containing samples (Figure 3a) show a triplet
characteristic of nitroxide radicals, originated by the hyperfine interaction between the unpaired electron
spin to the 14N nitrogen (S=1) in a fast motion regime. The observed lines could be satisfactorily
simulated (red curves in Figure 3) by considering axial A- and g-tensors, with g
= 12 MHz, A = 98 MHz, and isotropic Brownian rotational diffusion. Best-fit simulations were
obtained for rotational correlation times of 0.5±0.2 ns for the 3-carboxyl-PROXYL, and of the order of
ns for the amino acid building-blocks. The presence of EPR activity demonstrates that the nitroxyl
spin label stays active after all reaction steps of the building-block synthesis. Within the margin of error
±5%) the intensity of the EPR spectra, measured as area under the integrated spectra, of all three
⊥
= 2.0071, g = 2.0026,
║
A
⊥
║
1
(
building blocks is the same as the intensity observed for the precursor 05. This confirms the quantitative
functionalization of the building blocks.
7
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10.1002/cphc.201900211
Figure 3: Experimental (black curves) and simulated (red curves) EPR spectra of the final building-blocks for each amino
acid and for the 3-carboxyl-PROXYL precursor 05; (Hyp: hydroxyproline 21; Tyr: tyrosine 23; Ser: serine 22) measured at
98 K. Experimental conditions were identical for all samples. Samples were measured at concentrations of 1 mM in TCE.
For the simulations axial symmetry was considered for the A- and g-tensors, with g = 2.0071, g = 2.0026, A = 12 MHz,
= 98 MHz. The simulations also consider isotropic Brownian rotational diffusion, with rotational correlation times of
.5 ± 0.2 ns (3-carboxyl-PROXYL), 1.2 ± 0.2 ns (Hyp) and 1.3 ± 0.2 ns (Tyr, Ser). Convolution with a Lorentzian line with
2
⊥
║
⊥
A
║
0
peak-to-peak width of 0.1 mT was performed in order to reproduce the observed linewidths.
1
Figure 4: H MAS spectra recorded at 8 kHz spinning with background suppression with (mw on) and without microwave
(
mw off) irradiation for 15 60 mM of the building-block radical in TCE for a) Fmoc-NH-Hyp(OOC-PROXYL)-OH 21, b)
Fmoc-NH-Tyr(OOC-PROXYL)-OH 23 and c) Fmoc-NH-Ser(OOC-PROXYL)-OH 22. Spectra are normalized by the
maximum intensity of the mw off spectra in order to better show the different enhancement factors. Asterisks indicate
spinning sidebands.
8
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Figure 5: H → 13C CP MAS NMR spectra recorded at 8 kHz with (mw on) and without microwave (mw off) irradiation
for 60 mM of the building-block radical in TCE for a) Fmoc-NH-Hyp(OOC-PROXYL)-OH 21, b) Fmoc-NH-Tyr(OOC-
PROXYL)-OH 23 and c) Fmoc-NH-Ser(OOC-PROXYL)-OH 22. Spectra are normalized by the maximum intensity of the
mw off spectra in order to figure out the different enhancement factors.
1
1
1
13
H MAS and H → C CP MAS spectra for the paramagnetic building-blocks 21, 22, 23 were recorded
to inspect the efficiency of the synthesized building-blocks for DNP applications. The samples were
prepared as 60 mM TCE solutions, a concentration where the best DNP enhancement was found for
1
1
13
Fmoc-NH-Hyp(OOC-PROXYL)-OH 21 in H MAS and H → C CP MAS experiments (see Figures
1
S27 and S28 in the supplementary material). In the H MAS spectra (Figure 4), a signal is visible at
around 6 ppm for all three samples, which clearly refers to the isotropic signal of the solvent TCE.
1
Comparing the H MAS spectra (Figure 4 a,b,c) with (mw on) and without (mw off) microwave
irradiation, it is obtained that the signals in the MW on spectra are enhanced by factors of 25.9 for the
hydroxyproline, 16.6 for the tyrosine and 7.8 for the serine building-block (Table 1) compared to the
1
3
MW off spectra. Very similar signal enhancements (Table 1) are also observed in the C CP MAS
spectra of these samples for the signal at 70 ppm of the carbons of TCE (Figure 5). This observation is
1
consistent with a homogeneous polarization of all H nuclei in the TCE matrix, which transfer their
1
3
53
polarization uniformly to the C nuclei in the CP experiment. Thus, all three building-blocks exhibit
DNP enhancement.
Table 1: Enhancement factors  of 15mM a) Fmoc-NH-Hyp(OOC-PROXYL)-OH 21, b) Fmoc-NH-Ser(OOC- PROXYL)-OH
1
1
13
2
2 and c) Fmoc-NH-Tyr(OOC-PROXYL)-OH 23 in TCE measured at 8 kHz for H MAS, H → CP MAS and C MAS direct
channel resonances.
Hyp (21)
25.9 ± 0.1
24 ± 1
Ser (22)
16.6 ± 0.1
16 ± 1
Tyr (23)
7.8 ± 0.1
7 ± 1
¹
H MAS
1
H → ¹³C CP MAS
³C MAS (direct channel)
¹
12 ± 1
9 ± 1
7 ± 1
9
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Figure 6: 13C spectra recorded at 8 kHz spinning of 15 mM of the building-block in TCE for a) Fmoc-NH-Hyp(OOC-
1
3
PROXYL)-OH 21, b) Fmoc-NH-Tyr(OOC- PROXYL)-OH 23 and c) Fmoc-NH-Ser(OOC- PROXYL)-OH 22. (I) C MAS
1
3
spectra recorded with microwave (MW on) irradiation, (II) Direct channel C MAS spectra recorded with microwave (MW
1
3
1
on) irradiation using the modified C MAS pulse sequence where 180° pulses are employed on the H channel during build-
up of the C magnetization, (III) Indirect channel spectrum calculated as difference of (I) and (II), and (IV) 13C MAS spectra
13
recorded without microwave (MW off) irradiation.
To quantify the DNP efficiency in 13C direct polarization experiments, ¹³C MAS experiments were
recorded employing the saturation recovery pulse sequence. Since next to the direct polarization transfer
to ¹³C (direct channel), an alternative polarization pathway may occur, where polarization is transferred
via the proton spin reservoir (indirect channel),5
4-58
additional experiments with a pulse sequence
1
57
containing rotor synchronized 180° pulses on the H channel were recorded. The spectra for the
different ¹³C MAS experiments are shown in Figure 6. As also found in other systems, the vicinity of
1
3
the radicals broadens the line-width of the C-signals in the direct channel, compared to the line-width
in the indirect channel.54-57
13
The enhancement for C MAS direct polarization (direct channel) was extracted by comparing the mw
on spectra in Figure 6 II and the mw off spectra in Figure 6 IV. Enhancement factors of 12±1, 9±1 and
7
±1 (Table 1) were obtained for the hydroxyproline, tyrosine and serine building blocks, respectively.
1
1
These enhancements are smaller compared to the enhancements observed in the H and H → ¹³C CP
MAS experiments. A feasible explanation is the field dependence of the C enhancement, which may
differ from the H enhancement (see for example ). The detailed investigation of this field dependency
or an optimization of the transfer conditions and in particular the polarization field is however beyond
the scope of the present work. Comparing the three building blocks, the best enhancements are obtained
1
3
1
54
1
1
for the hydroxyproline (21) with enhancements of 25.9, 24, and 12 for H → ¹³C CP MAS, H MAS and
¹³C MAS (direct channel) respectively.
The obtained H and 13C-enhancement factors are one order of magnitude smaller than the typical
1
enhancements obtainable by “DNP juice” biradical solutions under favorite conditions. However, the
“
DNP juice” approach does not permit site-selective spin labelling. Comparing the enhancements
obtained by our approach to the previously reported ones employing a tethered biradical (TOTAPOL),2
1,
2
2, 25
which allow site-labelling only on the C-terminus, it is evident that our approach is more versatile
with respect to the position of the label and the enhancements obtained by us are of the same size or
exceed them.
1
0
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2.3 EPR- and DNP activity of the spin-labels in a synthesized peptide
In the next step the stability and the EPR- and DNP activity of the building-blocks inside an SPPS-
synthesized peptide has to be validated. For this, the five-residue peptide with the sequence Acetyl-Gly-
Ser(OOC-PROXYL)-Gly-Gly-Gly 24 was synthesized as a simple model-peptide using a standard SPPS
protocol59, 60 on a chlortrityl resin, by using the synthesized building-block the Fmoc-NH-Ser(OOC-
PROXYL)-OH 22 in the same way as a standard Fmoc-protected amino acid. The peptide cleavage from
the resin was performed using strongly acidic conditions of 95% TFA, which is the most common
condition for the cleavage step. The peptide was analyzed and purified by HPLC (S25). In the ESI-MS
+
spectra (S26) the calculated mass of the peptide 24 ([M+2H] = 545.3 m/z) was found as well as the
+
mass of the ionized peptide dimer at [2M+3H] = 1090.6 m/z, which clearly states the successful
synthesis and incorporation of the PROXYLated building-block 22 into the peptide sequence.
Figure 7: Experimental X-band EPR spectra of the synthesized peptide Acetyl-Gly-Ser(OOC-PROXYL)-Gly-Gly-Gly 24
in water (top) and used hydroxyproline Fmoc-NH-Ser(OOC-PROXYL)-OH 22 building-block in TEC (bottom) measured
at 298 K. Spectra were normalized by the maximum intensity. Experimental conditions were identical for all two samples.
Samples were measured at concentrations of 15 mM at ambient pH.
Figure (figure 7) compares the X-band EPR spectra of the neat building block 22 and the model peptide
2
4 in an aqueous solution. As is evident from the spectra, both samples exhibit the same hyperfine
coupling profile. This proves not only the EPR activity of the building block inside a peptide, but also
verifies the stability of the introduced spin label during SPPS and peptide cleavage from the resin.
1
1
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ChemPhysChem
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1
Figure 8: a) H MAS Spectra of 15 mM Acetyl-Gly-Ser(OOC-PROXYL)-Gly-Gly-Gly 24 in a glycerol-d8/D
2
O/H
with (MW on) and without (MW off) microwave irradiation measured at 8 kHz spinning. b) H → ¹³C CP MAS spectra of
5mM Acetyl-Gly-Ser(OOC-PROXYL)-Gly-Gly-Gly 24 in a glycerol-d8/D O/H O matrix measured with MW on/off.
2
O matrix
1
1
2
2
Note: The small signal at 0 ppm is attributed to the silicon plug, used to tightly close the liquid in the NMR rotor. Asterisks
indicate the spinning side bands of the glycerol.
Since the concentration of real biomolecule samples usually contains lower concentrations then 60 mM,
a smaller peptide concentration of 15 mM was employed to show the capability of the spin label, even
at lower concentrations. Figure 8 validates the DNP activity of the spin label inside the model peptide
1
1
13
2
4 by comparing the H MAS and H → C CP MAS spectra of the synthesized peptide 24 with and
1
without micro-wave irradiation. The H MAS spectra (Figure 8a) show a signal at around 6 ppm which
clearly refers to the signal of the used glycerol/water matrix. Comparing the H MAS spectra with (MW
1
on) and without (MW off) microwave irradiation, it is evident that the signals in the MW on spectra are
1
3
enhanced by factors of 12.1±0.1. Looking at the C CP MAS spectra, two signals at 60 and 70 ppm are
observed, which refer to the carbons of the glycerol-d8. For these signals, similar enhancements of
1
3.9±0.5 are observed. The enhancements obtained for the peptide 24 are even higher than those of the
corresponding building-block 22 at 15 mM concentration. These are excellent enhancements for mono
radical systems used in spin labeling,61-63 and even compared to other approaches that reached
enhancements between 10 and 27 by using biradical systems, which normally tend to result in drastically
higher enhancements.22, 25 Since many factors such as the matrix and the structure6, 55, 64 of the radical
itself strongly influence the enhancements, this change is not very surprising. Finally, it can be clearly
stated that the synthesized peptide was successfully and specifically labeled with our synthesized
building-block. EPR activity as well as signal enhancement in DNP NMR measurements is clearly
demonstrated. Moreover, the spin-labeling with mono-radicals is an efficient way for creating samples
for DEER/PELDOR experiments, e.g. in structural biochemistry or biophysics. This shows the big
potential of the presented strategy for their use in biologically relevant peptides.
1
2
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ChemPhysChem
10.1002/cphc.201900211
3
Experimental Section
3.1
Synthesis
3.1.1 General
Chemicals were purchased from Acros, Sigma-Aldrich, Carl Roth and Iris Biochem. Chemicals were
used without further purification unless explicitly mentioned.
3.2
Characterization
3.2.1 Liquid NMR
Solution-state NMR measurements have been conducted using a Bruker ARX 500 spectrometer
(1
H-NMR 500.16 MHz, C NMR 125.78 MHz) at the service department of the TU Darmstadt. The
13
chemical shifts are given in ppm. As internal standard the shift of the solvent peak was used.
3.2.2 Reverse phase high-performance liquid chromatography (HPLC)
Reversed phase high-performance liquid chromatography (RP-HPLC) for analytical analysis was
conducted using a Water HPLC Setup consisting a Water Alliance e2695 equipped with a Water 2998
PDA detector. The detection wave length was chosen depending on the analyte between 214, 254, 280
and 301 nm. The eluent system for the HPLC system comprised eluent A (0.1% aq. TFA) and eluent B
(99.9% acetonitrile containing 0.1% TFA). Unless otherwise specified analytical HPLC runs were
conducted at a flow rate of 2 ml/min with an eluent gradient from 20% to 80% of eluent A over 10 min.
For building-block analysis a RP 18 HP column from CS Chromatographie (3 μm, 120 Å) was used.
For peptide analysis a Nucleosil 100-5 C18 column from Macerey-Nagel (5µm, 100 Å) was used.
Preparative purification of the peptide was performed on a Knauer Multokrom RP18 column
2
3
0x250 mm (5 μm, 100 Å) employing a flow rate of 8 mL/min and a ACN gradient from 2% to 20% in
3 min.
3.2.3 Mass spectrometry (MS)
ESI (electron spray ionization) mass spectra were recorded with a Bruker Esquire-LC mass spectrometer
at the service department of the TU Darmstadt.
3.2.4 Electron paramagnetic resonance (EPR)
The EPR spectra for all samples were measured on an EPR Miniscope MS-400 (Magnettech, Germany)
equipped with an A TC H03 temperature controller and a rectangular TE102 resonator operating at
9
.43 GHz. All EPR spectra were recorded with a modulation amplitude of 0.1 mT and a modulation
frequency of 100 kHz. Unless otherwise specified spectra were taken under 30 dB of microwave
attenuation with a mantissa gain of 1 and an exponential gain of 1. Measurements were performed at
2
98 K. A magnetic field range of 25 mT was swept with a center B field of 337 mT with a sweeping
0
1
3
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ChemPhysChem
10.1002/cphc.201900211
time of 120 s to acquire 2048 points. The spectra of the peptide were measured directly after
chromatographic purification under acidic conditions without adjusting the pH to activate all radical
sites. Simulations of the EPR spectra were performed by assuming a fast-motional regime, using the
6
5
function “garlic” from the EasySpin® simulation package in MATLAB (MathWorks, Inc).
3.2.5 Dynamic nuclear polarization (DNP) NMR experiments
1
A 3.2 mm triple resonance H/X/Y low-temperature MAS probe was used to carry out the solid-state
DNP experiments at a Bruker Avance III 400 MHz NMR spectrometer. The spectrometer was equipped
with an AscendTM 400 sweep-able DNP magnet and a 9.7 T Bruker gyrotron system generating a µWave
1
1
13
frequency of 263 GHz. A MAS frequency of 8 kHz was employed for all H MAS, H → C CP MAS
(
(
1
3
cross polarization magic angle spinning), and C MAS NMR spectra. A sample temperature of 107 K
without µW) and 117 K (with µW) was nominally noted, where the µWave irradiation is the cause for
higher sample temperature at fixed maximum cooling power stabilized by a Bruker BioSpin MAS low
temperature cooling system. All H MAS, H → 13C CP MAS and 13C MAS NMR spectra were
1
1
measured once without microwave irradiation (MW off) and afterwards with the same parameters with
1
13
microwave irradiation (MW on). The frequencies were 400.02 MHz for H and 100.59 MHz for C,
1
respectively. For H MAS spectra a background suppression pulse sequence was applied where two
1
80° refocusing pulses after a 90° excitation pulse were employed with a spacing of 0.1 µs between the
pulses. The pulse lengths for the 90° excitation pulses were set to 2.58 µs and 16 scans were used. The
repetition delay was set to three times T of the protons, which was 7 s for Fmoc-NH-Hyp(OOC-
1
PROXYL)-OH 21, 4 s for Fmoc-NH-Ser(OOC-PROXYL)-OH 22 and 5 s for Fmoc-NH-Tyr(OOC-
1
PROXYL)-OH 23. To measure the proton T
1
times, H saturation recovery experiments were performed,
where variable delays for 12 data points varying from 0.01 s to 240 s were employed. The calculation
1
13
for the T
were measured with 16 scans, a repetition delay of three times T
000 µs. SPINAL-64 was used for decoupling.66 All building-blocks have been measured at a
concentration of 60mM in TCE and the peptide 24 at a concentration of 15mM in glycerol-8d/H O/D
v/v/v).
1
times were done using the Bruker topspin software package. H → C CP MAS NMR spectra
1
of the protons, and a contact time of
2
2
2
O
(
1
3
Direct polarization C MAS experiments were performed as saturation recovery experiments with and
without suppression of the indirect channel. In the saturation recovery experiments with indirect channel
5
7
suppression a string of rotor synchronized 180° pulses of 5.15 µs on the proton channel was applied
1
during the build-up time to suppress the build-up of H-magnetization and thus to suppress indirect
channel resonances in the spectra. The initial saturation pulse train to quench the thermal 13C
magnetization consists of twenty 2.2 µs pulses with 5 ms pulse spacing. A build-up time of 650 s was
employed. Spectra were recorded with 16 scans and SPINAL-64 decoupling was applied during data
acquisition.66
To analyze the signal enhancements, the spectra were fitted with a Lorentz function. Using the data of
the fits, calculation of the enhancement factors was conducted by dividing the peak heights from the
spectra with MW irradiation (IMWon) through the corresponding spectra without MW irradiation (IMWoff).
The respective deviations for the enhancement Δε were estimated according to eq. 1. The enhancement
ε is multiplied by the sum of the noise intensity to signal intensity ratio from the MW on and MW off
spectra. The noise intensities (noiseMWon and noiseMWoff) used for the calculations with eq. 1 are the
maximum levels of noise determined individually for the MW on and MW off spectrum, and IMWon and
I
MWoff are the absolute intensities of the signals, determined by the Lorentz fit, of the MW on and MW
off spectrum. Other influences caused by for example depolarization effects67-70 were not considered in
this calculation.
1
4
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ChemPhysChem
10.1002/cphc.201900211
noiseMWꢀꢁ
IMWꢀꢁ
noiseMWꢀff
IMWꢀff
Δε = ε ⋅ (
+
)
(1)
3.3
tBu protection of the hydroxyl group of Hyp, Ser and Tyr
t
3
.3.1 Synthesis of Fmoc-NH-Hyp(OH)-O Bu 10
tBu protection of the Fmoc-amino acids has been performed by adapted syntheses from literature.42, 43
Fmoc-NH-Hyp(OH)-OH 13 (2.88 g, 8.0 mmol, 1.00 equiv) was dissolved in 40 ml THF. After cooling
to 0°C tert-Butyl 2,2,2-trichloroacetimidate 09 (1.98 ml, 10.7 mmol, 1.33 equiv) was added slowly. The
next two hours, every hour two additions of 09 (each 1.98 ml, 10.7 mmol, 1.33 equiv) followed. The
reaction mixture was stirred at 60°C for 4 d. One final addition of 09 (0.75 ml, 4.0 mmol, 0.50 equiv)
and 2 h of reaction time resulted in full conversion of the substrate 13 as proved by HPLC. THF was
removed under reduced pressure and the obtained solid redissolved in DCM. The insoluble solid was
filtered off and the DCM filtrate was washed with a saturated aqueous solution of NaHCO
3
, 0.1 M
NaOHaq, distilled water and sat. aq. NaCl solution (brine). The solution was dried over Na SO , filtered,
2
4
and the solvent removed under reduced pressure to give 3.67 g of crude 10 as colorless solid. The
product was used for the next reaction steps without further purification.
1
0
+
1
HPLC: t
00 MHz): δ[ppm] = 7.89, 7.65, 7.42, 7.34, 4.29, 4.26, 4.14, 3.39, 1.90, 1.38 ppm.; C-NMR (DMSOP-
d6, 500 MHz): δ[ppm] = 162.81, 153.88, 143.72, 140.68, 127.63, 127.05, 125.04, 120.09, 80.47, 67.63,
6.59, 58.49, 54.97, 46.51, 37.85, 27.26 ppm.
R
= 6.26 min; ESI-MS: [M+H] = 410.2 m/z (calc. 410.2 m/z); H-NMR (DMSO-d6,
13
5
6
t
3
.3.2 Synthesis of Fmoc-NH-Ser(OH)-O Bu 11
t
According to the hydroxyproline Bu protection procedure, Fmoc-NH-Ser(OH)-OH 14 (2.67 g,
.0 mmol, 1.00 equiv) was dissolved in 40 ml of a 1:1 mixture of ethyl acetate and DCM. After cooling
8
to 0°C tert-Butyl 2,2,2-trichloroacetimidate 09 (1.98 ml, 10.7 mmol, 1.33 equiv) was added slowly. The
next two hours, every hour two additions of 09 (each 1.98 ml, 10.7 mmol, 1.33 equiv) followed. The
reaction mixture was stirred at RT for 4 d. One final addition of 09 (0.35 ml, 2.0 mmol, 0.25 equiv) and
2
h of reaction time resulted in full conversion of the substrate 14 as proved by HPLC. The ethyl
acetate/DCM mixture was removed under reduced pressure and the solid redissolved in DCM. The
insoluble solid was filtered off and the DCM filtrate was washed with a saturated aqueous solution of
NaHCO
3
, 0.1 M NaOHaq, distilled water and brine. The solution was dried over Na
2
SO , filtered, and
4
the solvent removed under reduced pressure to give 4.32 g of crude 11 as colorless solid. The product
was used for the next reaction steps without further purification.
1
1
1
5
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ChemPhysChem
10.1002/cphc.201900211
+
1
HPLC: t = 6.17 min; ESI-MS: [M+H] = 384.19 m/z (calc. 384.18 m/z); H-NMR (DMSO-d6,
5
R
13
00 MHz): δ[ppm] = 7.88, 7.73, 7.42, 7.33, 6.92, 4.29, 4.22, 4.01, 3.50, 1.39 ppm.; C-NMR (DMSOP-
d6, 500 MHz): δ[ppm] = 162.81, 155.98, 143.76, 140.67, 127.58, 127.00, 25.20, 120.04, 80.53, 65.65,
1.34, 57.24, 46.60, 27.66 ppm.
6
t
3
.3.3 Synthesis of Fmoc-NH-Tyr(OH)-O Bu 12
t
According to the hydroxyproline Bu protection procedure, Fmoc-NH-Tyr(OH)-OH 15 (0.79 g,
2
2
.0 mmol, 1.00 equiv) was dissolved in 20 ml ethyl acetate. The next three hours, every hour tert-butyl
,2,2-trichloroacetimidate 09 (each 0.49 ml, 2.7 mmol, 1.33 equiv) was added at RT. The reaction
mixture was stirred at RT for 3 d. One addition of 09 (0.20 ml, 1.0 mmol, 0.5 equiv) followed. After
2
0
4 h the reaction mixture was heated to 40°C. Two more additions of 09 (each 0.20 ml, 1.0 mmol,
.5 equiv) followed. The solution was stirred for 2 h to result in full conversion of the substrate 15 which
was proved by HPLC. Ethyl acetate was removed under reduced pressure and the solid was redissolved
in DCM. The insoluble solid was filtered off and the DCM filtrate was washed with a saturated aqueous
solution of NaHCO
3
, 0.1 M NaOHaq, distilled water and brine. The solution was dried over Na
2
SO ,
4
filtered, and the solvent removed under reduced pressure to give 1.16 g crude 12 as colorless solid. The
product was used for the next reaction steps without further purification.
1
2
+
+
HPLC: t
R
= 8.07 min; ESI-MS: [M+H] = 460.21 m/z (calc. 460.2 m/z), [(M)
2
+H] = 919.42 m/z (calc.
1
9
4
1
19.42 m/z); H-NMR (DMSO-d6, 500 MHz): δ[ppm] = 8.99, 7.87, 7.67, 7.41, 7.34, 7.03, 6.68, 4.23,
.18, 4.05, 2.80, 1.53 ppm.; 13C-NMR (DMSOP-d6, 500 MHz): δ[ppm] = 171.07, 155.78, 143.70,
40.64, 129.99, 127.20, 121.29, 119.93, 114.89, 65.56, 56.35, 46.58, 35.95, 26.89 ppm.
3.4
Nitroxide coupling to the side chain
t
3
.4.1 Synthesis of Fmoc-NH-Hyp(OOC-PROXYL)-O Bu 16
PROXYLation by esterification reaction has been performed by an adapted synthesis from literature.44
t
Fmoc-NH-Hyp(OH)-O Bu 10 (1135 mg, 2.40 mmol, 1.00 equiv) was dissolved in 23 ml DCM. 1-ethyl-
3
1
-(3-dimethylaminopropyl)carbodiimid hydrochloride 19 (EDC×HCl) (510 mg, 2.64 mmol,
.10 equiv), 4-Dimethylaminopyridine 20 (DMAP) (33 mg, 0.26 mmol, 0.11 equiv) and 3-Carboxy-
PROXYL 05 (502 g, 2.64 mmol, 1.10 equiv) were added. The solution was stirred at RT for 12 h. The
reaction progress was monitored by HPLC. After reaction was completed the solution was washed with
0
.5 M HClaq, a saturated aqueous solution of NaHCO
3
and brine. The solution was dried over Na
2
SO ,
4
filtered, and the solvent removed under reduced pressure to give 16 (1.26 g, 90.9%) as a yellow solid.
1
6
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ChemPhysChem
10.1002/cphc.201900211
1
6
+
+
HPLC: t
R
= 9.40 min; ESI-MS: [M+H] = 577.3 m/z (calc. 577.3 m/z), [M+Na] = 600.3 m/z (calc.
6
00.3 m/z)
t
3
.4.2 Synthesis of Fmoc-NH-Ser(OOC-PROXYL)-O Bu 17
t
According to the hydroxyproline esterification reaction procedure, Fmoc-NH-Ser(OH)-O Bu 11
215 mg, 0.50 mmol, 1.00 equiv), 19 (EDC×HCl) (107 mg, 0.55 mmol, 1.10 equiv),
-dimethylaminopyridine 20 (DMAP) (7 mg, 0.06 mmol, 0.11 equiv) and 3-carboxy-PROXYL 05
105 mg, 0.55 mmol, 1.10 equiv) give 180 mg (65.3%) of crude 17 as yellow solid.
(
4
(
1
7
+
+
HPLC: t
5
R
= 9.04 min; ESI-MS: [M+H] = 551.3 m/z (calc. 551.3 m/z), [M+H] = 552.3 m/z (calc.
52.3 m/z), [M+2H] = 553.3 m/z (calc. 553.3 m/z)
+
t
3
.4.3 Synthesis of Fmoc-NH-Tyr(OOC-PROXYL)-O Bu 18
t
According to the hydroxyproline esterification reaction procedure Fmoc-NH-Tyr(OH)-O Bu 12
278 mg, 0.50 mmol, 1.00 equiv), 19 (EDC×HCl) (105 mg, 0.55 mmol, 1.10 equiv),
-Dimethylaminopyridine 20 (DMAP) (7 mg, 0.06 mmol, 0.11 equiv) and 3-Carboxy-PROXYL 05
107 mg, 0.55 mmol, 1.10 equiv) give 227 mg (74%) of crude 12 as yellow solid.
(
4
(
1
8
+
+
HPLC: t
6
R
= 7.82 min; ESI-MS: [M] = 627.3 m/z (calc. 627.3 m/z), [M+H] = 628.3 m/z (calc.
28.3 m/z), [M+2H] = 629.3 m/z (calc. 629.3 m/z)
+
1
7
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ChemPhysChem
10.1002/cphc.201900211
3.5
tBu deprotection to restore the carboxyl group
3.5.1 Synthesis of Fmoc-NH-Hyp(OOC-PROXYL)-OH 21
4
5
Deprotection reaction has been performed according to adapted syntheses from literature. Fmoc-NH-
t
Hyp(OOC-PROXYL)-O Bu 16 (1.26 g, 2.2 mmol, 1.00 equiv) was dissolved in 14 ml DCM and cooled
to 0°C. While cooling TFA (15.63 ml, 194.0 mmol, 89.00 equiv) was added, which turns the solutions
black. The solution was allowed to warm up to RT and the solvent was removed under reduced pressure
to dryness. The residual solid was redissolved in DCM and washed three times with a small amount of
distilled water and brine (DCM:washing solution= 3:1). The solution was dried over Na
2
SO , filtered,
4
and the solvent removed under reduced pressure. The residue was purified by column chromatography
(n-hexane: Ethyl acetate:Acetic acid = 47.5:47.5:5) to give 21 (0.54 g, 47%) as a yellow solid.
2
1
+
HPLC: t = 5.47 min; ESI-MS: [M] = 521.2 m/z (calc. 521.2 m/z), [M+H]+= 522.2 m/z (calc.
R
5
22.2 m/z), M+2H]+= 523.2 m/z (calc. 523.2 m/z)
3.5.2 Synthesis of Fmoc-NH-Ser(OOC-PROXYL)-OH 22
t
t
According to the hydroxyproline Bu deprotection procedure Fmoc-NH-Ser(OOC-PROXYL)-O Bu 17
(0.18 g, 0.3 mmol, 1.00 equiv) and TFA (2.33 ml, 28.9 mmol, 89.00 equiv) in 2 ml DCM give 100 mg
crude 22 as yellow solid, which was purified by column chromatography (n-hexane:Ethyl acetate:Acetic
acid = 1:1) to give 22 (0.04 g, 24%) as a yellow solid.
2
2
+
+
HPLC: t
4
m/z),[M+NH
R
= 5.44 min; ESI-MS:[M] = 496.21 m/z (calc. 496.21 m/z), [M+H] = 497.21 m/z (calc.
97.21 m/z), [M+2H] = 498.21 m/z (calc. 498.21 m/z),[M+Na] = 518.20 m/z (calc. 518.20
+
+
]+= 513.20m/z (calc. 513.20 m/z)
4
3.5.3 Synthesis of Fmoc-NH-Tyr(OOC-PROXYL)-OH 23
t
t
According to the hydroxyproline Bu deprotection procedure Fmoc-NH-Tyr(OOC-PROXYL)-O Bu 18
(0.23 g, 0.4 mmol, 1.00 equiv) and TFA (2.65 ml, 32.9 mmol, 89.00 equiv) in 2.4 ml DCM give 150 mg
1
8
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ChemPhysChem
10.1002/cphc.201900211
crude 18 as yellow solid, which was purified by column chromatography (n-hexane: Ethyl acetate:
Acetic acid = 47.5:47.5:5) to give 23 (0.12 g, 58%) as a yellow solid.
2
3
+
+
+
HPLC: t
5
R
= 6.30 min; ESI-MS:[M] = [M] = 571.2 m/z (calc. 571.2 m/z), [M+H] = 572.2 m/z (calc.
72.2 m/z), [M+2H] = 573.2 m/z (calc. 573.2 m/z)
+
3.6
Synthesis of Acetyl-Gly-Ser(OOC-PROXYL)-Gly-Gly-Gly 24 peptide
Peptide synthesis has been performed according to adapted syntheses from literature.59 Pre-dried
chlortrityl resin (0.3 g, 0.46 mmol, 1.00 equiv, maximum loading 1.52 mmol/g) was placed in a 10 mL
synthetic syringe with frit bottom and swollen in DMF for 30 min. The resin is pre-activated with 2.5
ml of a diisopropylethylamine (DIEA) / dimethylformamide (DMF) mixture (v/v = 1/7) for 60 min.
Amino acid coupling: Each amino acid coupling was carried out twice before Fmoc cleavage. A
coupling time of 30 min was used for each coupling step. After each coupling step the resin is washed
4
0
times with DMF. The first amino acid was coupled to the activated resin using Fmoc-Gly-OH (0.27 g,
.91 mmol, 2.00 equiv) and DIEA (0.24 g, 1.82 mmol, 4.00 equiv) in 1.7 mL DMF. For the second and
third
-[bis(Dimethylamin)methylen]-1H-1,2,3-triazol[4,5-b]pyridinium-3-oxid-hexafluorophosphat
HATU) (0.34 g, 0.90 mmol, 1.98 equiv) and DIEA (0.24 g, 1.82 mmol, 4.00 equiv) in 1.7 mL DMF
were used. Building-block coupling was performed using Fmoc-NH-Ser(OOC-PROXYL)-OH 22
0.29 g, 0.57 mmol, 1.25 equiv), HATU (0.21 g, 0.56 mmol, 1.23 equiv) and DIEA (0.15 g, 1.14 mmol,
double
coupling
step
Fmoc-Gly-OH
(0.27 g,
0.91 mmol,
2.00 equiv),
1
(
(
2
0
4
.50 equiv) in 1.2 mL DMF. The last double coupling step was performed using Ac-Gly-OH (0.11 g,
.91 mmol, 2.00 equiv), HATU (0.34 g, 0.90 mmol, 1.98 equiv) and DIEA (0.24 g, 1.82 mmol,
.00 equiv) in 1.9 mL DMF.
Fmoc cleavage: Fmoc deprotections was performed after each amino acid double coupling with a
solution of 20% (v/v) piperidine in DMF for 5 min and in a second step for 15 min. After the second
deprotection step the resin was washed 6 times with DMF before the next amino acid is coupled. Ac-
Gly-OH coupling is not followed by a deprotections step.
Peptide cleavage: Before peptide cleavage the loaded resin was washed 6 times with DCM and dried
over night at high vacuum. For cleavage 3 mL of cleavage solution (95 % trifluoroacetic acid / anisole
/
triisopropylsilane = 95/2.5/2.5 (v/v/v) is used over the cleavage time of 3 h. The peptide was
precipitated in cold Methyl tert-butyl ether (MTBE), followed by an incubation at -20 °C for 30 minutes.
The precipitated peptide was washed once with MTBE and once with diethyl ether to give spin labeled
peptide 24 (113 mg, 46%) as a white solid. After preparative purification by HPLC and basic workup to
remove TFA 43 mg (17%) of spin labeled peptide 24 was obtained with a purity (HPLC) of >95%.
1
9
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ChemPhysChem
10.1002/cphc.201900211
2
4
+
+
HPLC: t
R
= 8.29 min; ESI-MS:[M+2H] = 545.3 m/z (calc. 545.3 m/z), [2M+3H] = 1089.5 m/z
(calc.1089.5 m/z).
4
Conclusion
The amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) were spin labeled by PROXYL
at their hydroxyl side chain in three reaction steps. The synthesized building-blocks showed activity of
the nitroxyl species in electron paramagnetic resonance (EPR) measurements. DNP enhancements of
TCE solutions of up to 25.9 for protons and carbons were obtained using a spin label concentration of
6
0 mM. The final building-blocks were N-terminally protected by a Fmoc-protection group and C-
terminally accessible for coupling reactions. The presented approach allowed the synthesis of side chain
spin labeled amino acids that show EPR activity and chemical stability even under the harsh acidic
conditions occurring in SPPS. This was demonstrated by the synthesis of a small peptide starting from
one of the presented building-blocks and showing spin label activity by EPR and DNP NMR
1
13
measurements. In the latter enhancement factors of 12.1±0.1 for H and 13.9±0.5 for C were obtained
using a peptide concentration of 15 mM. This will allow the use of these building-blocks in solid phase
peptide synthesis to create specifically spin labeled peptides or mini-proteins for ligation studies
employing DEER or PELDOR or to create peptide-based radicals for DNP NMR applications in future.
Finally, this universal spin labeling strategy is available for a wide variety of amino acids, which provide
a hydroxyl group at their sidechain.
Acknowledgements:
Financial support by the Deutsche Forschungsgemeinschaft in the framework of grants Bu-911/24-1 is
gratefully acknowledged. Dr. Marcos de Oliveira Jr. thanks for financial support by the Alexander from
Humboldt Research Foundation.
2
0
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ChemPhysChem
10.1002/cphc.201900211
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[
[
4] M. Renault, A. Cukkemane, M. Baldus Angew. Chem. Int. Ed. 2010, 49, 8346-8357.
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TOC:
TOC Text:
SPPS Spin-Labeling: A versatile strategy for the spin-labeling of peptides by non-natural amino-acids
with a nitroxide-radical in their side-chain is presented. This strategy is fully compatible to solid phase
peptide synthesis (SPPS). The success of the labeling and the activity of the radical is demonstrated by
EPR and DNP enhanced solid-state-NMR measurements of a small model peptide.
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