Sonogashira, CuAAC, and oxime ligations for the synthesis of MnI tricarbonyl photocorm peptide conjugates

Article abstract of DOI:10.1002/ejic.201402123

In this work, facial tricarbonylmanganese(I) complexes [Mn(bpea ^CH2C6H4R)(CO)₃]PF₆ incorporating a functionalized 2,2-bis(pyrazolyl)ethylamine (bpea) ligand with R = I, C≡CH, and CHO have been explored for their utility in bioorthogonal coupling to carrier peptides bearing N-terminal alkyne, azide, and aminoxy residues. As a model system, the transforming growth factor β-recognizing (TGF-β) peptide sequence R'-Leu-Pro-Leu-Gly-Asn-Ser-His-OH was used in which R' is the reactive group complementary to the metal complex functionality. The use of catalyst-free oxime ligation gave the most stable conjugate with no degradation observed by HPLC over 96 h even after repeated freeze-thaw cycles. Both the parent complex as well as the functionalized peptide were investigated for photoactivated CO delivery to heme proteins by using the myoglobin assay and found to have essentially identical release properties. This work has established a new strategy for the conjugation of photoactivatable CO-releasing molecules (PhotoCORMs) to biological carrier systems. Copyright

Full text of DOI:10.1002/ejic.201402123

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DOI:10.1002/ejic.201402123
Sonogashira, CuAAC, and Oxime Ligations for the
Synthesis of MnI Tricarbonyl PhotoCORM Peptide
Conjugates
Sandesh Pai,[a] Krzysztof Radacki,[a] and Ulrich Schatzschneider*[a]
Keywords: Bioinorganic chemistry / CO release / Bioorthogonal coupling / Peptides / Carbonyl ligands / Manganese
In this work, facial tricarbonylmanganese(I) complexes
[Mn(bpeaCH2C6H4R)(CO)3]PF6 incorporating a functionalized
2,2-bis(pyrazolyl)ethylamine (bpea) ligand with R = I, CϵCH,
and CHO have been explored for their utility in bioortho-
gonal coupling to carrier peptides bearing N-terminal alk-
yne, azide, and aminoxy residues. As a model system, the
transforming growth factor β-recognizing (TGF-β) peptide
sequence RЈ-Leu-Pro-Leu-Gly-Asn-Ser-His-OH was used in
which RЈ is the reactive group complementary to the metal
complex functionality. The use of catalyst-free oxime ligation
gave the most stable conjugate with no degradation ob-
served by HPLC over 96 h even after repeated freeze–thaw
cycles. Both the parent complex as well as the functionalized
peptide were investigated for photoactivated CO delivery to
heme proteins by using the myoglobin assay and found to
have essentially identical release properties. This work has
established a new strategy for the conjugation of photoacti-
vatable CO-releasing molecules (PhotoCORMs) to biological
carrier systems.
also include the action of esterase and phosphatase enzymes
on carbonylmetal complexes (enzyme-triggered CORMs,
ET-CORMs),[13–16] the use of magnetothermal heating in
an alternating electrical field,[17] and the use of light to stim-
ulate the liberation of CO (PhotoCORMs).[18–21] Only more
recently the focus of the field has shifted from the simple
introduction of an ever-increasing number of novel metal
carbonyl core structures as CORMs to a proper design of
the whole system to really turn these compounds into viable
drug candidates. As nicely outlined by Romao et al., in ad-
dition to the so-called inner CORM sphere, which controls
the stoichiometry and kinetics of CO release, special atten-
tion must also be paid to the drug sphere, the outer ligand
periphery of a carbonylmetal complex, which determines its
distribution and uptake in different tissues and cells.[22] By
very careful selection of functionalized isocyanato ligands
coordinated to a molybdenum(0) tricarbonyl core, for ex-
ample, it has been possible to achieve very selective liver
accumulation to alleviate the toxic effects of acetamino-
phen-induced severe acute liver injury.[23] As an alternative
to the design of small-molecule CO prodrugs, the conjuga-
tion of established CORMs to macromolecules as well as
hard and soft nanomaterials for the targeted delivery of car-
bon monoxide has also been explored. In addition to CO-
and CORM-loaded micelles and metal–organic frameworks
(MOFs),[24,25] carbon- and silica-based nanoparticles have
also been decorated with CO-releasing molecules by using
the CuAAC click reaction.[26,27] A particular focus has been
on carrier peptides for the targeted delivery of CORMs,[28]
because solid-phase peptide synthesis (SPPS) allows a facile
variation of the amino acid sequence and thus modulation
Introduction
It is now well established that carbon monoxide, endoge-
nously produced by the action of heme oxygenase (HO) en-
zymes on heme,[1] has a very important biological function
in higher organisms, including humans, in particular in the
context of small-molecule signaling and protection against
oxidative stress.[2–5] During the last decade, methods to in-
tervene in these CO-modulated biochemical processes for
potential therapeutic applications in human medicine have
gained steadily increasing attention. Whereas the controlled
application of CO gas by inhalation is of limited use only
due to the fixed partition ratio between lungs, blood, and
different tissues, making a selective targeting of specific dis-
eased structures in the body difficult, organic and organo-
metallic compounds have been developed as prodrug carrier
systems for carbon monoxide, for which the term “CO-re-
leasing molecules” (CORMs) was coined by Motterlini and
co-workers.[6–11] A wide range of metal carbonyl co-ligand
structures have been explored for their utility as CORMs,
and different trigger mechanisms are now well established
to initiate the release of CO from the metal coordination
sphere. In addition to simple ligand exchange reactions with
solvent or the constituents of biological systems,[12] they
[a] Institut für Anorganische Chemie, Julius-Maximilians-
Universität Würzburg,
Am Hubland, 97074 Würzburg, Germany
E-mail: ulrich.schatzschneider@uni-wuerzburg.de
http://www-anorganik.chemie.uni-wuerzburg.de/en/research/
prof_dr_u_schatzschneider
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201402123.
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of the cellular and tissue accumulation. In addition to cell- ary amines, giving very stable pendant functionalization.
penetrating peptides,[29] which are generally capable of Although the 4-iodobenzyl and 4-ethynylbenzyl bpea li-
membrane transition, there are also peptides that are more gands required for Sonogashira and CuAAC coupling have
specifically recognized by complementary binding partners. already been reported in previous work,[46] the correspond-
Although robust organometallic half-sandwich complexes ing aldehyde-functionalized ligand was still missing from
can be directly conjugated to a growing peptide chain dur- our arsenal. To prevent the formation of 1,4-disubstituted
ing SPPS by amide bond formation,[30–36] most CORMs are byproducts and allow selective reduction of the imine in the
not stable under the standard conditions for cleavage from presence of the aldehyde group, bpeaNHCH2C6H4CHO (5) was
the solid support and thus require a post-labeling strategy synthesized starting from 2,2-bis(pyrazolyl)ethylamine (1),
by bioorthogonal coupling reactions.[37–40] So far, our work which was first condensed with 4-(diethoxymethyl)benzal-
has mostly relied on palladium-mediated Sonogashira cou- dehyde (2) and then, without isolation of the imine 3, re-
pling and copper-catalyzed azide–alkyne cycloaddition duced with sodium borohydride in methanol to give 4
(CuAAC) reactions,[26,27,41] which are, however, limited in (Scheme 1). Removal of the acetal protective group with 2 m
scope due to the use of a potentially toxic transition-metal hydrochloric acid gave the desired ligand 5 in 70% yield as
catalyst. Thus, in this paper, we report on the facile synthe- a yellow oil. Reaction of 5 with pentacarbonylmanganese
sis of a novel aldehyde-functionalized tridentate ligand and bromide in acetone at reflux under nitrogen and the ex-
the use of its tricarbonylmanganese(I) complex in oxime lig- clusion of light then afforded the corresponding facial
ation to an aminoxy-functionalized transforming growth tricarbonylmanganese(I) complex 6 in 80% yield as a yel-
factor β-recognizing (TGF-β) peptide.[42] This very mild low solid upon precipitation with aqueous potassium hexa-
and catalyst-free conjugation method[43–45] is contrasted fluorophosphate. The two related complexes [Mn-
with more conventional Sonogashira- and CuAAC-based (bpeaN=CHC6H4I)(CO)3]PF6 (7) and [Mn(bpeaN=CHC6H4CϵCH)-
procedures and is shown to give excellent results in terms (CO)3]PF6 (8), incorporating an iodo- or alkyne-function-
of conjugate accessibility and stability.
alized bpea ligand, respectively, were available by a similar
procedure described in previous work.[46]
1
The H and 13C NMR as well as IR spectra of the new
Results and Discussion
ligand 5 are in full accordance with the expected structure.
The ATR IR spectrum of complex 6 shows two strong
bands at 2036 and 1928 cm–1, as expected for C3v symmetry,
Synthesis and Spectroscopic Characterization
We recently introduced the 2,2-bis(pyrazolyl)ethylamine assigned to symmetrical and antisymmetrical CϵO stretch-
(bpea) ligand into the field of CO-releasing molecules ing vibrations, respectively. An additional peak at 1691 cm–1
(CORMs) due to its facile functionalization at the primary is due to the C=O vibrational band of the aldehyde func-
amino group by condensation with substituted aldehydes. tional group (Figure 1). In addition, coordination to the
In particular, benzaldehyde derivatives give rise to quite metal center leads to a shift in the methine proton signal in
stable imine adducts, which can be further converted by so- the 1H NMR spectrum from δ = 6.55 to 7.47 ppm. Further-
dium borohydride reduction into the corresponding second- more, the methylene protons at the α position with respect
Scheme 1. Synthesis of [Mn(bpeaNHCH2C6H4CHO)(CO)3]PF6 (6) for catalyst-free oxime ligation.
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to the coordinated amine group now each appear as a CO ligands in a facial arrangement with the three nitrogen
2
doublet at δ = 3.57 and 3.18 ppm with J coupling con- donor groups from the bpea ligand completing the octahe-
stants of 13.2 and 13.8 Hz, respectively, indicative of the dral coordination environment and the 4-formylbenzyl
diastereotopic environment generated by metal complex- group pointing away from the metal center. Although the
ation. The signals are both further split by coupling with distances between the Mn center and the two pyrazolyl N
the adjacent methine and NH protons (see Figure S1 in the donor atoms of 2.032(2) and 2.047(2) Å are shorter by
Supporting Information). The positive-mode ESI mass about 0.11 Å than the third manganese–amine N bond
spectrum shows one signal at m/z = 434.08, which is as- length of 2.151(2) Å, the Mn–C and CϵO bond lengths
signed to the cationic [M – PF6]+ unit of complex 6, and show essentially no variation with the nature of the trans N
an additional one at m/z = 350.09 due to the species [M – ligand.
3 CO – PF6]+ (see Figure S2).
Figure 2. Molecular structure of the cationic unit of 6 with thermal
ellipsoids drawn at the 50% probability level. The hexafluorophos-
phate counter ion is not shown for clarity. Selected bond lengths
Figure 1. ATR IR spectrum of [Mn(bpeaNHCH2C6H4CHO)(CO)3]PF6
(6).
[Å] and angles [°]: Mn–C(2) 1.813(3), Mn–C(4) 1.817(3), Mn–C(6)
1.811(3), Mn–N(8) 2.151(2), Mn–N(12) 2.032(2), Mn–N(17)
2.047(2), C(2)–O(3) 1.147(4), C(4)–O(5) 1.146(4), C(6)–O(7)
1.141(4), C(28)–O(29) 1.219(4); C(2)–Mn–C(6) 88.22(14), C(4)–
Mn–C(6) 90.24(14), C(2)–Mn–C(4) 87.87(13), N(12)–Mn–N(17)
87.29(9), N(8)–Mn–N(12) 84.95(9), N(8)–Mn–N(17) 82.70(9),
C(6)–Mn–N(8) 91.68(12), C(2)–Mn–N(8) 174.31(12), C(4)–Mn–
N(8) 97.82(12), C(6)–Mn–N(12) 176.57(12), C(2)–Mn–N(12)
95.20(11), C(4)–Mn–N(12) 89.63(11), C(6)–Mn–N(17) 92.89(13),
C(2)–Mn–N(17) 91.62(12), C(4)–Mn–N(17) 176.82(11).
X-ray Crystallography
Crystals of 6 suitable for X-ray structure analysis were
obtained by recrystallization from dichloromethane/diethyl
ether at room temperature. The crystallographic parameters
are summarized in Table 1, and the molecular structure is
shown in Figure 2. The compound crystallizes in the mono-
clinic space group P21/n. The MnI center is bound to three
Bioorthogonal Conjugation and Characterization of Peptide
Conjugates
Table 1. Crystallographic parameters for complex 6.
For the synthesis of the PhotoCORM peptide conju-
gates, three different bioorthogonal coupling methods were
evaluated. In all cases the amino acid sequence RЈ-
LPLGNSH-OH was used[47] derived from a TGF β1-bind-
ing peptide with RЈ being a suitable N-terminal building
block for the coupling with the bpea ligand bearing the
complementary coupling partner. Whereas metal complex
7 – incorporating an iodobenzyl group on the bpea ligand –
was to be used in a Sonogashira reaction, the alkyne-func-
tionalized analogue 8 is suitable for CuAAC coupling. In
addition, new aldehyde-modified ligand 5 and its manga-
nese(I) complex 6 were synthesized for oxime ligation to the
peptide.[45] The peptide sequence was assembled by solid-
phase peptide synthesis (SPPS) on a preloaded H-l-
His(Trt)-2CT resin by using standard Fmoc methodology.
Then, N-terminal functionalization with 5-hexynoic acid, 2-
azidoacetic acid, or (aminooxy)acetic acid was carried out
on the resin using identical methodology, followed by con-
Empirical formula
Mr
Dimensions [mm]
Space group
a [Å]
b [Å]
c [Å]
C19H17F6MnN5O4P
579.29
0.14ϫ0.12ϫ0.05
P21/n
15.0215(14)
9.2082(11)
16.4618(14)
91.226(5)
2276.5(4)
4
1.690
100(2)
0.737
0.71073
27.50
β [°]
V [Å3]
Z
ρcalcd. [gcm–3]
T [K]
μ [mm–1]
λ [Å] (Mo-Kα)
2θmax [°]
Reflections measured
Unique reflections [IϾ2σ(I)]
Variables
4492
3560
307
0.0424
R1[a] (I Ն 2σ)
wR2[b] (I Ն 2σ)
0.0972
[a] R1 = Σ||Fo| – |Fc||/Σ|Fo|. [b] wR2 = {Σ[w(Fo – Fc2)2]/Σ[w(Fo ) ]}1/2
.
2
2 2
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Scheme 2. Synthesis of peptide conjugates 13 and 14 by CuAAC and catalyst-free oxime ligation.
jugation to 6–8. The peptides were cleaved from the solid peptide (see Figure S5). However, it proved difficult to iso-
support with a cocktail of TFA/TIS/H2O (90:5:5), with TIS late the modified peptide in high purity, because repeated
= triisopropylsilane, and purified by preparative HPLC preparative HPLC never gave a completely pure product.
when necessary to give peptides 9–11 (Scheme 2). The pept-
ides were obtained in high purity (97%) and in yields of
55–60%. Then Sonogashira coupling of 7 with the hexynoic
acid terminated peptide 9 was investigated under different
standard conditions by using [PdCl2(PPh3)2] as catalyst and
copper(I) iodide as co-catalyst.[48–50] However, despite re-
peated attempts with different modifications of the reaction
conditions, no conjugate 12 could be detected in any case,
as monitored by ESI-MS and HPLC. Therefore, we turned
to the CuAAC “click” reaction between alkyne-function-
alized complex 8 and the azidoacetic acid modified peptide
10, which was carried out in DMF/water (1:1, v/v) using
copper(II) sulfate as the catalyst and sodium ascorbate as
the in situ reductant to generate the active copper(I) species
(Scheme 2). The resulting product was analyzed by HPLC
and showed one main peak with a retention time of
30.1 min, which is significantly longer than that of peptide
10, for which tR = 22.9 min (Figure 3). Further proof of the
Figure 3. Analytical HPLC chromatogram of peptide 10 (black,
220 nm) and conjugate 13 (blue, 350 nm) after purification by pre-
parative HPLC.
Therefore, we analyzed the stability of conjugate 13 by
successful CuAAC coupling, which leads to a triazole link- analytical HPLC in acetonitrile/water (10:90, v/v). In ad-
age between the metal complex and the peptide, came from dition to the initial major peak at tR = 30.1 min, a second
ESI-MS. For peptide 10, a major peak is observed at m/z signal with a slightly shorter retention time of 27.6 min
= 820.40, which is due to [M + H]+ (see Figure S3 in the gradually grew in and reached an intensity of 30% relative
Supporting Information). Conjugate 13, on the other hand, to the major peak after 39 h (Figure 4). This slow decompo-
shows one peak arising from a monocationic species at m/z sition of the conjugate is presumably due to hydrolysis of
= 1247.45, assigned to [M – PF6]+, in addition to peaks the imine bond in the bpea part of the ligand under these
from dicationic species at m/z = 624.23 and 582.24, due to conditions.
[M – PF6 + H]2+ and [M – 3 CO – PF6 + H]2+, respectively
Therefore, as an alternative method, we turned to oxime
(see Figure S4). The ATR IR spectrum of conjugate 13 also ligation, which proceeds under mild and catalyst-free condi-
shows two bands at 2040 and 1932 cm–1, indicative of the tions to give very stable conjugation products and also miti-
incorporation of the intact tricarbonylmetal moiety into the gates problems of purification due to co-reactants. In ad-
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Peptide conjugate 14 was obtained in moderate yield (58%)
but with an excellent purity of 98%. Analytical HPLC of
the product showed a significantly longer retention time
with respect to peptide 11 with tR = 28.3 min. In the ESI
mass spectrum, two main signals from monocationic species
are found at m/z = 1225.45 and 435.65, which correspond
to the conjugate [M – PF6]+ and a fragment peak resulting
from cleavage of the oxime linkage assigned to [Mn-
(bpeaCHO)(CO)3 + H]+, respectively (Figure 6). The suc-
cessful conjugation of the complex to the peptide is also
evident from the two strong CϵO vibrational bands in the
IR spectrum at 2042 and 1935 cm–1 (see Figure S6 in the
Supporting Information).
Figure 4. Normalized HPLC traces (350 nm detection) of peptide
conjugate 13 upon incubation in acetonitrile/water (10:90, v/v) re-
corded at room temperature over 39 h.
dition, the bpea ligand was modified to incorporate a more
stable secondary amine as in 6 instead of the imine linkage
in 8 between the bis(pyrazolyl) moiety and the rest of the
ligand (Scheme 1). For the coupling, (aminooxy)acetic acid
(Aoa) was attached to the N-terminus of the peptide se-
quence through solid-phase peptide synthesis (SPPS), as de-
scribed earlier (Scheme 2).[44] Two repeated couplings with
10 equiv. of Fmoc-Aoa-OH were required to give a negative
Kaiser test. Peptide 11 was obtained as a white solid in 60%
yield after purification by preparative HPLC. Analytical
HPLC showed a high purity (97%) of the product with a
retention time of 15.6 min. Further characterization by
ESI-MS gave only a single major peak at m/z = 810.41,
assigned to [M + H]+ (Figure 5).
Figure 6. Positive-mode ESI mass spectrum of conjugate 14 and
(inset) overlay of analytical HPLC chromatograms of metal com-
plex 6 (blue, 350 nm), peptide 11 (black, 220 nm), and peptide con-
jugate 14 (red, 350 nm).
Stability Studies in DMSO and Water
To gain an insight into the stability of the novel (bpea)
tricarbonylmanganese(I) complex 6 and its peptide conju-
gate 14, UV/Vis spectroscopy studies were carried out. Ow-
ing to the poor water solubility of complex 6, it was dis-
solved in dimethyl sulfoxide. No spectral changes could be
observed over a period of 16 h when the compound was
kept in the dark. Illumination at 365 nm with a UV hand
lamp, coincident with the MLCT absorption maximum of
the Mn(bpea)(CO)3 moiety, however, led to a quick fading
of the main absorption band at 357 nm (Figure 7 and Fig-
ure S7 in the Supporting Information). The stability of the
peptide conjugate 14, on the other hand, was more conve-
niently investigated by using RP-HPLC in pure water.
HPLC traces were recorded after repeated freeze–thaw cy-
cles (–25 °C to room temperature) under the exclusion of
Figure 5. ESI+ mass spectrum and analytical HPLC chromatogram
(inset) of peptide 11 (220 nm detection).
For the conjugation, Aoa-functionalized peptide 11 was light and showed no signals other than the main peak of
treated with [Mn(bpeaNHCH2C6H4CHO)(CO)3]PF6 (6) in a the conjugate at tR = 28.3 min, which could be observed
mixture of tetrahydrofuran/phosphate buffer (1:1, v/v) at unaltered over the course of 4 d (Figure 8) and indicates the
pH 5.0 at room temperature for 3 h (Scheme 2). The mix- excellent dark stability of both the Mn(CO)3 moiety with
ture was desalted on a short reversed-phase column (C18- the reduced amine ligand 5 as well as the oxime linkage to
SepPak) and subsequently purified by preparative HPLC. the peptide.
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spectrum (Figure 9 and Figures S8 and S9 in the Support-
ing Information), which indicates that both the parent com-
plex and the conjugate are stable under these experimental
conditions. Thus, both compounds act as dark-stable pro-
drugs for phototriggered CO release. Only upon illumina-
tion at 365 nm were characteristic changes in the UV/Vis
spectra noted that are indicative of the conversion of
MbFeII (deoxy-Mb) with λmax = 557 nm into MbFeIICO
(carboxy-Mb) with λmax = 540 and 577 nm (see Figure S10).
The concentration of the MbFeIICO formed was calculated
by using
a molar extinction coefficient of ε540 =
15.4 mm–1 L–1).[6] Approximately 2 equiv. of CO were re-
leased per molecule of either 6 or 14 (Figure 9 and Fig-
ures S11 and S12). The CO release data with the half-lives
of both the complex and the conjugate are presented in
Table 2. Differences in the CO release parameters between
both compounds are small and probably due to difficulties
in the accurate preparation of stock solutions of the peptide
conjugate due to its highly hygroscopic nature.
Figure 7. Changes in the absorbance of 6 at 357 nm upon incu-
bation in dimethyl sulfoxide in the dark for up to 16 h and during
subsequent photolysis of the same solution with an UV hand lamp
at 365 nm. See Figure S7 for the full spectral traces.
Figure 9. Change in absorption at 540 nm showing the stability of
complex 6 (15 μm) in the dark (over the course of 16 h) and the
formation of MbCO upon illumination at 365 nm in 0.1 m PBS at
pH 7.4 in the presence of myoglobin (60 μm) and sodium dithionite
(10 mm) under nitrogen.
Figure 8. Normalized HPLC traces (350 nm detection) of peptide
conjugate 14 upon incubation in water for a period of up to 96 h.
The conjugate was dissolved in water, and HPLC traces were re-
corded after repeated freeze–thaw cycles (–25 °C to room tempera-
ture) under the exclusion of light.
CO Release Measurements by the Myoglobin Assay
Quantum Yield Determination
The standard myoglobin assay was used to compare the
CO release behavior of both the parent complex 6 and
Standard ferrioxalate actinometry was used to determine
the peptide conjugate 14, exploiting the conversion of de- the quantum yield of the CO release reaction. The quantum
oxy-Mb into carbonmonoxy-Mb.[51,52] A freshly prepared yields (Φ) for both the complex and the conjugate are listed
buffered aqueous solution of horse skeletal myoglobin in Table 2 and are in the order of 10–4. These Φ values were
(MbFeII) was reduced with sodium dithionite solution un- calculated from the initial spectral changes and are much
der nitrogen. Incubation in the dark for up to 16 h under lower than those of other carbonylmetal complexes such
the conditions of the myoglobin assay did not lead to any as Na3[W(CO)5(TPPTS)][53] and [Mn(tpa)(CO)3]ClO4.[54,55]
noticeable changes in the Q-band region of the UV/Vis Nonetheless, it should be taken into account that these ex-
Table 2. CO release data and quantum yields at 365 nm (Φ365) for 6 and 14.
kCO [10–4 s–1]
Φ365 [10–4]
[a]
[b]
[c]
Compound
[MbCO] [μm]
CO released [equiv.]
t1/2 [min]
6
14
30.71Ϯ0.40
25.05Ϯ0.01
2.06Ϯ0.03
1.70Ϯ0.08
22.58Ϯ0.33
15.51Ϯ0.52
5.5
7.4
4.36Ϯ0.03
4.52Ϯ0.14
[a] Under the conditions of myoglobin assay. [b] In DMSO solution from UV/Vis spectra. [c] Calculated by using a photon flux of the
UV hand lamp (2.82Ϯ0.05)ϫ10–8 Einsteins–1.
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3000 instrument equipped with a diode array detector and a Re-
proSil 100 column (C18, 5 μm, 4.6 mm for analytical and 10 mm
diameter for preparative HPLC, 250 mm length) using a linear gra-
dient of 5–90% acetonitrile/water containing 0.1% TFA as the elu-
ent over 40 min at a flow rate of 0.6 mLmin–1 for analytical and
3.0 mLmin–1 for preparative chromatography. The myoglobin assay
and all photophysical studies were carried out in a quartz cuvette
(d = 1 cm) with an Agilent 8453 UV/Vis diode array spectropho-
tometer.
periments were performed under the conditions of the myo-
globin assay rather than in pure solvent with the protein
showing a significant internal shielding effect at the exci-
tation wavelength of 365 nm.[56,57]
Conclusions
In this work we used the facile functionalization of the
bpea ligand framework to introduce three different groups
into the periphery of the complex that are suitable for bio-
orthogonal coupling reactions, namely iodine for Sonoga-
shira coupling with alkyne-terminated peptides, an alkyne
for the CuAAC click reaction with azidoacetic acid modi-
fied peptides, and a p-benzaldehyde group for oxime lig-
ation with (aminooxy)acetic acid functionalized systems.
Under the different conditions examined, we could not ob-
tain a coupling product by the Sonogashira reaction with
complex 7, but complexes 6 and 8 gave the corresponding
carbonylmetal complex peptide conjugates linked through
either an oxime or triazole linker. It turned out that not
only the coupling methodology is vital for successful conju-
gation, but also the bpea ligand structure. Whereas the
imine-based ligand showed slow hydrolysis in acetonitrile/
water over the course of 1–2 d, the corresponding amine
compound, when combined with the oxime linker, was
highly stable for up to 96 h, even under repeated freeze–
thaw cycles. Both the parent complex and the peptide con-
jugate are CO prodrugs when kept in the dark, but release
up to 2 equiv. of carbon monoxide upon photoactivation.
Thus, this work has allowed the facile, catalyst-free prepara-
tion of CORM-peptide conjugates for the targeted delivery
of carbon monoxide to biological systems.
4-({[2,2-Bis(pyrazolyl)ethyl]amino}methyl)benzaldehyde
(bpeaNHCH2C6H4CHO, 5): 4-(Diethoxymethyl)benzaldehyde (2;
0.58 g, 2.82 mmol) was added to a solution of [2,2-bis(pyrazolyl)-
ethyl]amine (1; 0.50 g, 2.82 mmol) in anhydrous methanol (20 mL).
The mixture was heated at reflux in the presence of molecular sieves
(4 Å) under nitrogen for 40 h. The molecular sieves were then fil-
tered off, and the solution was cooled to 0 °C to give the intermedi-
ate imine 3. Solid sodium borohydride (186.70 mg, 4.94 mmol) was
added to reduce the imine 3, and stirring was continued for 7 h at
room temperature. Water (10 mL) was added to the reaction mix-
ture and the product extracted with dichloromethane (100 mL) and
washed with brine (3ϫ 50 mL). The organic layer was separated,
dried with magnesium sulfate (ca. 10 g), and filtered. The solvent
was then removed under reduced pressure to give the intermediate
aminoacetal 4 as a yellow oil. Yield: 57% (0.60 g, 1.60 mmol).
Aminoacetal 4 (0.59 g, 1.60 mmol) was treated with 2 m hydro-
chloric acid (8 mL) and the mixture stirred at room temperature
for 3 h. The reaction mixture turned curdy white, was made alk-
aline with aqueous sodium hydroxide, and extracted with dichloro-
methane (3ϫ 50 mL). The combined organic extracts were washed
with brine (3ϫ 100 mL), dried with sodium sulfate (ca. 10 g), and
filtered. The solvent was then removed under reduced pressure to
give the product 5 as a yellow oil. Yield: 70% (0.33 g, 1.12 mmol).
IR (ATR): ν = 3115 (m, NH), 2844, 1691 (s, C=O), 1606, 1390,
˜
1
1090 cm–1. H NMR (300 MHz, CDCl3): δ = 9.98 (s, 1 H, CHO),
3
3
7.82 (d, J = 8.3 Hz, 2 H, 2,6-HPh), 7.57 (d, J = 2.4 Hz, 2 H, 3-
3
3
Hpz), 7.55 (d, J = 1.7 Hz, 2 H, 5-Hpz), 7.43 (d, J = 7.9 Hz, 2 H,
3
3
4
3,5-HPh), 6.55 [t, J = 6.9 Hz, 1 H, CH(pz)2], 6.28 (dd, J = 2.4, J
= 1.8 Hz, 2 H, 4-Hpz), 3.90 (s, 2 H, CH2C6H4) 3.69 (d, 3J = 6.9 Hz,
2 H, CH2-CH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 51.21
(CH2CH), 53.03 (CH2C6H4), 75.12 [C(pz)2], 106.92 (C-4pz), 128.55
(C-3,5Ph), 129.14 (C-2,6Ph), 130.13 (C-5pz), 135.66 (C-1Ph), 140.49
(C-3pz), 147.04 (C-4Ph), 192.07 (CHO) ppm. MS (ESI+, MeOH):
m/z = 318.13 [M + Na]+. C16H17N5O (295.35): C 65.06, H 5.80, N
23.71; found C 64.82, H 5.75, N 23.28.
Experimental Section
General: Reactions were carried out in oven-dried Schlenk glass-
ware under pure nitrogen when necessary. Solvents were dried with
molecular sieves and degassed prior to use. All reagents were pur-
chased either from Sigma–Aldrich or Alfa Aesar and used without
further purification. Pentacarbonylmanganese bromide was ob-
tained from Strem Chemicals. 2,2Ј-Bis(pyrazol-1-yl)ethylamine [Mn(bpeaNHCH2C6H4CHO)(CO)3]PF6 (6): Pentacarbonylmanganese
(bpea),[58] [Mn(bpeaN=CHC6H4I)(CO)3]PF6 (7),[46] and [Mn-
(bpeaN=CHC6H4CϵCH)(CO)3]PF6 (8)[46] were prepared according to
published procedures. Azidoacetic acid and Fmoc-(aminooxy)ace-
tic acid were synthesized according to literature methods.[44,59,60]
NMR spectra were recorded with a Bruker DRX 300 spectrometer
(1H: 300.13 MHz; 13C: 75.47 MHz) at ambient temperature. Chem-
ical shifts (δ) are given in ppm downfield from tetramethylsilane
(TMS) and are referenced to the solvent signal.[61] Coupling con-
stants J are given in Hz. Individual peaks are marked as singlet (s),
doublet (d), doublet of doublet (dd), triplet (t), or multiplet (m).
Mass spectra were recorded with a Bruker MicroTOF (ESI) instru-
ment; only characteristic fragments are given for the most abundant
isotope peak. Data were recorded in positive ion mode. IR spectra
were recorded as pure solid samples by using a Nicolet 380 FT-IR
Spectrometer equipped with a SMART iTR ATR unit. Elemental
analysis (C, H, N) was performed with an EA3000 Elemental Ana-
lyser from HEKAtech GmbH, Wegberg. HPLC analysis of the
peptides and conjugates was performed with a Dionex Ultimate
bromide (368 mg, 1.34 mmol) and bpeaNHCH2C6H4CHO (5; 346 mg,
1.17 mmol) were dissolved in anhydrous acetone (50 mL) and
heated at reflux under nitrogen with the exclusion of light for 5 h.
The solvent was removed in vacuo, and the yellow residue was re-
dissolved in methanol (10 mL), and an aqueous solution of potas-
sium hexafluorophosphate (430.70 mg, 2.34 mmol) was added. The
yellow product that precipitated was filtered off, washed with water
and diethyl ether, and dried under vacuum. Yield: 80% (544.00 mg,
0.94 mmol). IR (ATR): ν = 3252 (m, N–H), 2036 (s, CϵO), 1928
˜
(s, CϵO), 1691 (s, CHO), 1607, 1412, 1288, 832 cm–1. 1H NMR
(300 MHz, [D6]acetone): δ = 10.04 (s, 1 H, CHO), 8.54 (d, 3J =
3
3
2.2 Hz, 1 H, 3Ј-Hpz), 8.43 (d, J = 2.2 Hz, 1 H, 3-Hpz), 8.34 (d, J
3
= 2.6 Hz, 1 H, 5Ј-Hpz), 8.28 (d, J = 2.7 Hz, 1 H, 5-Hpz), 7.93 (d,
3J = 8.3 Hz, 2 H, 3,5-HPh), 7.71 (d, 3J = 8.1 Hz, 2 H, 2,6-HPh),
7.47 [br. s, 1 H, CH(pz)2], 6.75 (t, 3J = 2.4 Hz, 1 H, 4-Hpz), 6.68 (t,
3J = 2.4 Hz, 1 H, 4Ј-Hpz), 5.77 (br. s, 1 H, NH), 4.83 (dd, 3J =
13.5, 4.1 Hz, 1 H, CH2C6H4), 4.38 (dd, 3J = 13.4, 10.3 Hz, 1 H,
CH2C6H4), 3.57 (ddd, 3J = 13.2, 7.9, 3.1 Hz, 1 H, CH2NCH2C6H4),
Eur. J. Inorg. Chem. 2014, 2886–2895
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3.18 (dd, 3J = 13.8, 8.9 Hz, 1 H, CH2NCH2C6H4) ppm. 13C NMR
(75.47 MHz, [D6]acetone): δ = 60.93 (CH2N), 72.23 (CH2C6H4),
79.28 [C(pz)2], 118.98 (C-4,4Јpz), 140.26 (C-3,5Ph), 140.92 (C-2,6Ph),
144.94 (C-5Јpz), 145.42 (C-5pz), 147.30 (C-4Ph), 152.73 (C-1Ph),
required to attain complete coupling, as indicated by a negative
Kaiser test. Cleavage time: 3 h with TFA/TIS/H2O (90:5:5, v/v).
The peptide was obtained as a white solid. Yield: 60% (185 mg,
0.23 mmol). RP-HPLC: tr = 15.60 min. MS (ESI+, MeOH): m/z =
157.68 (C-3pz), 157.81 (C-3Јpz), 202.23 (CHO) ppm. MS (ESI+, 810.41 [M + H]+. IR (ATR): ν = 3288 (m), 2961 (m), 1658 (s), 1537
˜
MeOH): m/z = 434.08 [M – PF6]+. C19H17F6MnN5O4P (579.28):
calcd. for C 39.39, H 2.95, N 12.09; found C 38.91, H 3.03, N
11.61.
(s), 1439 (m), 1198 (s), 1133 (s) cm–1.
[Mn(bpeaCϵC(CH2)3CO–TGFβ1–OH)(CO)3]PF6 (12): [Mn(bpeaN=CHC6H4I)-
(CO)3]PF6 (7; 24.30 mg, 36 μmol) and the peptide 5-hexynoic-
TGFβ1-OH (9; 30 mg, 36 μmol) were dissolved in dmf (1 mL) and
triethylamine (1.5 mL), and the solution was degassed by three
freeze–pump–thaw cycles. Copper(I) iodide (1 mg, 4.32 μmol,
12 mol-%) and cis-dichlorobis(triphenylphosphine)palladium(II)
(1 mg, 1.44 μmol, 4 mol-%) were added under nitrogen, and the
reaction mixture was degassed. The yellow solution was stirred at
room temperature under the exclusion of light for 28 h and then
loaded onto a short reversed-phase column (Waters C18 Sep-Pak,
5 g) washed with water (5ϫ 10 mL). However, no conjugate forma-
tion could be detected under these conditions as probed by HPLC
and ESI-MS.
General Conditions for the Solid-Phase Peptide Synthesis (SPPS):
The peptides were synthesized manually in a filter syringe on a
0.39 mmol scale according to the method described by Kirin et al.
using the Fmoc strategy.[62] A preloaded H-l-His(Trt)-2CT resin
was used as the solid support. The Fmoc-protected amino acids
were deprotected with a solution of 30% piperidine in dmf. For
each coupling step, Fmoc-amino acids (5 equiv., 0.97 m in dmf) and
coupling reagent (HOBT/HBTU, 0.97 m in dmf) were used. Diiso-
propylethylamine (DIEA; 10 equiv.) was used as the activator base.
The completeness of each coupling step was monitored by the Kai-
ser test.[63] The final coupling of 5-hexynoic acid, azidoacetic acid,
and (aminooxy)acetic acid was performed by using a solution of
5-hexynoic acid/HOBT/HBTU, azidoacetic acid/HOBT/HBTU or
Fmoc-(aminooxy)acetic acid/HOBT/HBTU in dmf with DIEA as
activator base over 2 h. The peptide was cleaved from the solid
support manually in a filter syringe at room temperature using a
cleavage cocktail of TFA/TIS/H2O (90:5:5). The peptide was then
isolated by precipitation with cold diethyl ether (–25 °C) and re-
peated cycles of washing, centrifuging, and decanting. The residue
was then dissolved in acetonitrile/water (1:1, v/v) and lyophilized
to yield the peptides as white solids.
[Mn(bpeaC 2 H N 3 – C H 2 C O – TG F β 1 – O H )(CO)3 ]PF6 (13): [Mn-
(bpeaN=CHC6H4CϵCH)(CO)3]PF6 (8; 28.02 mg, 48.90 μmol) and the
peptide N3-Ac-TGFβ1-OH (10; 40.10 mg, 48.90 μmol) were dis-
solved in dmf/water (1:1, v/v, 2 mL). Sodium ascorbate (0.70 mg,
3.50 μmol, 25 mol-%) and copper(II) sulfate pentahydrate
(0.15 mg, 0.70 μmol, 5 mol-%) were subsequently added, and the
mixture was stirred at room temperature under the exclusion of
light for 24 h. The yellow solution was loaded onto a short re-
versed-phase column (Waters C18-SepPak, 5 g) washed with water
(5ϫ 10 mL). The conjugate was then eluted with acetonitrile as a
HCϵC(CH2)3CO-Leu-Pro-Leu-Gly-Asn-Ser-His-OH (9): Peptide 9 yellow band. The solution was lyophilized to give the product as a
was synthesized on a 0.24 mmol scale on preloaded H-l-His-
yellow solid. Yield: 20% (13 mg, 9.50 μmol). RP-HPLC: tr
=
(Trt)-2CT resin (300 mg, 0.78 mmolg–1) by using the amino acids
30.10 min. MS (ESI+, MeOH): m/z = 1247.45 [M – PF6]+. IR
Fmoc-l-Ser(tBu)-OH, Fmoc-l-Asn(Trt)-OH, Fmoc-l-Gly-OH, (ATR): ν = 2958 (m), 2040 (s, CϵO), 1932 (s, CϵO), 1663 (s), 1533
˜
Fmoc-l-Leu-OH, Fmoc-l-Pro-OH, and Fmoc-l-Leu-OH under
manual solid-phase peptide synthesis conditions as described
above. Two repeated couplings of 5-hexynoic acid (10 equiv.) were
required to attain complete coupling, as indicated by a negative
Kaiser test. Cleavage time: 3 h with TFA/TIS/H2O (90:5:5, v/v).
The peptide was obtained as a white solid. Yield: 54% (106 mg,
0.13 mmol). RP-HPLC: tr = 22.80 min. MS (ESI+, MeOH): m/z =
(m), 1413 (m), 1132 (m) cm–1.
[Mn(bpeaNHCH2C6H4CH=OCH2CO–TGFβ1–OH)(CO)3]PF6 (14): Com-
plex [Mn(bpeaNHCH2C6H4CHO)(CO)3]PF6 (6; 5.79 mg, 0.01 mmol)
and the peptide Aoa-TGFβ1-OH (11; 10 mg, 0.01 mmol) were dis-
solved in a 1:1 (v/v) mixture of tetrahydrofuran (2 mL) and phos-
phate buffer saline (PBS; 2 mL, 100 mm, pH 5.2). The resulting yel-
low solution was stirred at room temperature under the exclusion
of light for 3 h. The solvent was removed by lyophilization, and
the yellow residue was dissolved in acetonitrile/water (1:9, v/v) and
loaded onto a short reversed-phase column (Waters C18-SepPak,
5 g) washed with water (5ϫ 10 mL) followed by acetonitrile (5ϫ
10 mL). The conjugate was then eluted with acetonitrile/water (1:1,
v/v) as a yellow band. The solution was lyophilized to give the
product as a yellow solid. Yield: 58% (8 mg, 0.006 mmol). RP-
HPLC: tr = 28.30 min. MS (ESI+, MeOH): m/z = 1225.45 [M –
831.43 [M + H]+. IR (ATR): ν = 3290 (m), 2959 (m), 1659 (s), 1623
˜
(s), 1538 (s), 1437 (m), 1197 (s), 1134 (s) cm–1.
N3-Ac-Leu-Pro-Leu-Gly-Asn-Ser-His-OH (N3-Ac-TGFβ1-OH, 10):
Peptide 10 was synthesized on a 0.39 mmol scale on preloaded H-
l-His(Trt)-2CT resin (500 mg, 0.78 mmolg–1) by using the amino
acids Fmoc-l-Ser(tBu)-OH, Fmoc-l-Asn(Trt)-OH, Fmoc-l-Gly-
OH, Fmoc-l-Leu-OH, Fmoc-l-Pro-OH, and Fmoc-l-Leu-OH un-
der manual solid-phase peptide synthesis conditions as described
above. Two repeated couplings of azidoacetic acid (10 equiv.) were
required to attain complete coupling, as indicated by a negative
Kaiser test. Cleavage time: 3 h with TFA/TIS/H2O (90:5:5, v/v).
The peptide was obtained as a white solid. Yield: 55% (172 mg,
0.21 mmol). RP-HPLC: tr = 22.90 min. MS (ESI+, MeOH): m/z =
PF ]+. IR (ATR): ν = 3284 (m), 2042 (s, CϵO), 1934 (s, CϵO),
˜
6
1662 (s), 1546 (s), 1413 (m), 1195 (s), 1132 (s) cm–1.
Myoglobin Assay:[51,52] In a quartz cuvette, a solution of horse skel-
etal muscle myoglobin (576 µL, 60 µm) in 0.1 m phosphate buffer
(PBS, pH 7.4) was degassed by bubbling with nitrogen and reduced
by the addition of sodium dithionite (10 mm, 100 μL) in PBS buffer
(0.1 m, pH 7.4) to a total volume of 676 μL for 6 and 795 μL for
820.40 [M + H]+. IR (ATR): ν = 3284 (m), 2958 (m), 2109 (s, N )
˜
3
1658 (s), 1536 (s), 1440 (m), 1200 (s), 1134 (s) cm–1.
Aoa-Leu-Pro-Leu-Gly-Asn-Ser-His-OH (Aoa-TGFβ1-OH, 11): 14. Compound 6 or 14 in dimethyl sulfoxide (10 μL, 15 µm each)
Peptide 11 was synthesized on a 0.319 mmol scale on preloaded H- was added to this solution, followed by PBS to give a total volume
l-His(Trt)-2CT resin (500 mg, 0.78 mmolg–1) by using the amino of 1000 μL with final concentrations of 15 μm of 6, 15 μm of 14,
acids Fmoc-l-Ser(tBu)-OH, Fmoc-l-Asn(Trt)-OH, Fmoc-l-Gly-
OH, Fmoc-l-Leu-OH, Fmoc-l-Pro-OH, and Fmoc-l-Leu-OH un-
der manual solid-phase peptide synthesis conditions as described
above. Two repeated couplings of Fmoc-Aoa-OH (10 equiv.) were
10 mm of sodium dithionite, and 60 μm of myoglobin with A557 Ͻ
1. The solutions were freshly prepared for both the dark-stability
measurements and the photoillumination experiments. Illumina-
tions were carried out under nitrogen at 365 nm with a UV hand
Eur. J. Inorg. Chem. 2014, 2886–2895
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FULL PAPER
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lamp (6 W, UVITEC, UK) positioned perpendicular to the cuvette
at a distance of 3 cm. Illuminations were interrupted at regular in-
tervals of 5 min for the initial 60 min and then at 10 min intervals
to record UV/Vis spectra with an Agilent 8453 UV/Vis diode array
spectrophotometer. As a dark control, measurements were carried
out by using the automated spectrometer software for a defined
period of time (16 h). All the illumination experiments were carried
out in triplicate.
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Ferrioxalate Actinometry: Ferrioxalate actinometry was used to de-
termine the photon flux of the 365 nm UV hand lamp because of
its sensitivity, wide spectral range, including the ultraviolet, and
ease of use.[64,65] The whole ferrioxalate actinometry procedure, in-
cluding the preparation of solutions, was carried out under red
safe-light. The number of moles of ferrous iron formed was deter-
mined spectrophotometrically by complexation with 1,10-phen-
anthroline (phen) to give the colored tris(phenanthroline) complex
[Fe(phen)3]2+ with λmax = 510 nm. In a 1 cm quartz cell, 0.006 m
(3 mL) of potassium ferrioxalate in 0.05 m sulfuric acid as the
chemical actinometer was irradiated with a 365 nm UV hand lamp
under efficient stirring. This irradiated solution (1 mL) was mixed
with 0.1% 1,10-phenanthroline in water and sodium acetate buffer
(0.5 mL) in water (1 m, pH 3.5) and further diluted to 10 mL with
water. A reference was prepared in the same way except that it was
not irradiated. Both solutions were placed in the dark (about 1 h)
to allow complexation to complete. The absorbance was then mea-
sured at 510 nm (ε = 11100 m–1 cm–1). A510 was maintained within
the range of 0.4–1.0. The photon flux of the 365 nm UV hand lamp
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was then calculated by using the following equation with Φ365 nm
=
1.21, V1 = volume of potassium ferrioxalate illuminated, V2 = vol-
ume of V1 used for complexation with 1,10-phenanthroline, V3 =
10–3ΔAV1V3
total volume:[66] Φp =
.
Φλε510V2t
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X-ray Crystallography: A single crystal of 6 was immersed in a film
of perfluoropolyether oil, mounted on a glass fiber, and transferred
to the diffractometer in a stream of cold nitrogen. Diffraction data
were collected with a Bruker D8 Quest diffractometer at 100 K
using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å).
Final cell constants were obtained from a least-squares fit of a sub-
set of a few thousand strong reflections. The APEX2 program (ver.
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drawing of the structures. CCDC-956477 contains the supplemen-
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) within the project FOR 630 “Biological function of organo-
metallic compounds”.
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Received: February 28, 2014
Published Online: May 16, 2014
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