Design and Synthesis of New Protease-Triggered CO-Releasing Peptide–Metal-Complex Conjugates

Article abstract of DOI:10.1002/ejoc.201901206

Aiming at the development of novel protease-triggered CO-releasing molecules (PT-CORMs) we designed a series of oxycyclohexadiene-Fe(CO)₃ complexes, which are connected through a self-immolative linker with a plasmin-specifying peptide unit. The challenging synthesis of these compounds was elaborated employing a combination of solid-phase peptide synthesis and solution chemical synthesis. Late-stage attachment of the organometallic unit and Pd-catalyzed cleavage of an alloc-protected lysine side chain afforded the target compounds in good yields and high purity.

Full text of DOI:10.1002/ejoc.201901206

DOI: 10.1002/ejoc.201901206
Communication
Peptide Conjugates | Very Important Paper |
Design and Synthesis of New Protease-Triggered CO-Releasing
Peptide–Metal-Complex Conjugates
Nikolay S. Sitnikov,[a,b] Yulia B. Malysheva,[b] Alexey Yu. Fedorov,*[b] and
Hans-Günther Schmalz*[a]
Abstract: Aiming at the development of novel protease-trig- elaborated employing a combination of solid-phase peptide
gered CO-releasing molecules (PT-CORMs) we designed a series synthesis and solution chemical synthesis. Late-stage attach-
of oxycyclohexadiene-Fe(CO)3 complexes, which are connected ment of the organometallic unit and Pd-catalyzed cleavage of
through a self-immolative linker with a plasmin-specifying pept- an alloc-protected lysine side chain afforded the target com-
ide unit. The challenging synthesis of these compounds was pounds in good yields and high purity.
Introduction
peutic agent requires the development of alternative methods
for its delivery. Carbon monoxide releasing molecules (CORMs)
are compounds capable to of delivering controlled amounts of
CO to the body without affecting the level of COHb produced.
They represent potential pharmaceutical agents of high clinical
value.
Carbon monoxide (CO), one of the three known gasotransmit-
ters, is endogenously produced in the human body through
heme oxygenase, which exists in three isoforms: the constitu-
tive HO-2 and HO-3 and inducible HO-1.[1] As CO has a high
affinity to hemoglobin (ca. 230-fold higher than that of oxygen)
it causes strong inhibition of O2 transport throughout the body
by formation of carboxyhemoglobin (COHb). This leads to
hypoxia and an overall intoxication of the organism.[2] However,
more recently it was revealed that CO actually represents an
essential signaling molecule[3] and its endogenous actions af-
fect the neuronal, cardiovascular, immune, respiratory, repro-
ductive, and gastrointestinal systems.[4] Thus, CO plays a vital
role in many physiological functions in the mammalian body
and exerts anti-inflammatory, antiapoptotic, anti-coagulative,
anti-hypertensive, antioxidant, and cell-protective effects.[5] In
principle, CO has great therapeutic potential against a wide
range of human diseases and pathological states including can-
cer, bacterial infections, inflammatory diseases, cardiovascular
disorders, rheumatoid arthritis, cerebral malaria, lung injury,
neurodegeneration, kidney and liver dysfunctions, chronic re-
jection after organ transplantation, and others.[6,7] However, a
person would need to inhale a comparably high concentration
of the gas to attain a meaningful concentration in the body.[5c]
This hampers the clinical use of carbon monoxide gas because
of the risk of intoxication.[5c] Therefore, the use of CO as a thera-
The first generation of CORMs, suggested by Motterlini,
mainly included metal carbonyl complexes (based on Cr, Mo,
Mn, Re, Fe, Ru) but also other types of compounds such as
“boranocarbonate” (CORM A1).[1b,3c,5d,6a] While these first-gener-
ation CORMs are stable and storable compounds, a disadvan-
tage is that they spontaneously release CO through ligand ex-
change upon dissolution in aqueous media.[1b] In contrast,
enzyme-triggered CO-releasing molecules (ET-CORMs) allow a
controlled intracellular CO delivery as they are activated only
by specific enzymes.[8] The first representatives of the ET-CORM
family were esterase-activated acyloxydiene tricarbonyl iron
complexes (Figure 1 and Scheme 1).[8a–8c] The concept was later
expanded to phosphatase- and penicillin G amidase-activated
analogs (Figure 1).[8d–8e] Other options to achieve a controlled
CO release[9] are based on pH-sensitive CORMs[10] or the use of
light[11] or heat[10,12] as external stimuli.
[a] Department of Chemistry, University of Cologne
Greinstrasse 4, 50939 Koeln, Germany
E-mail: Schmalz@uni-koeln.de
Figure 1. Structures of selected enzyme-triggered CORMs (ET-CORMs).
[b] Department of Organic Chemistry, Nizhny Novgorod State University
Gagarina Av. 23, 603950 Nizhny Novgorod, Russian Federation
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201901206.
© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. ·
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
ET-CORMs are activated by enzyme-induced hydrolysis to
form a highly labile dienol-Fe(CO)3 intermediate, which in turn
rapidly undergoes oxidation under physiological conditions to
liberate up to three molecules of CO as the main bioactive
product, besides a Fe3+ ion and cyclohexenone (Scheme 1). The
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biocompatibility, tissue selectivity and cell permeability[15a]
and tunable CO release kinetics.
)
Results and Discussion
Scheme 1. Mechanism of enzyme-triggered CO release from ET-CORMs.
Aiming at the development of novel PT-CORMs based on
plasmin as a specifier, we chose a general design in which the
plasmin specifier is linked to the CORM unit through a self-
immolative linker (Figure 2).
enzyme-triggered CO release from such complexes was proven
by means of the UV-based myoglobin assay and by headspace
GC analysis. Also, ET-CORMs showed very promising biological
effects in different cell-based assays.[13]
Today's knowledge about the construction of prodrugs with
controlled in vivo distribution and activation forms a solid basis
for the design of new organometallic CORMs for possible thera-
peutic applications.[14] In this context, protease-activated pro-
drugs are particularly promising since they allow drug delivery
to tissues with an enhanced expression of certain proteases.[15]
Indeed, modifications in proteolytic activity are associated with
diseases like cancer, as well as neurodegenerative and cardio-
vascular disorders.[16] Plasmin represents a serine protease that
plays a key role in tumor invasion and metastasis and is known
to be overexpressed in tumor tissues.[15a,17] It is essentially an
intracellular protease which is rapidly deactivated in the blood
circulation.[17] Increased plasmin activity is also strongly associ-
ated with rheumatoid arthritis (RA).[15a,18] Therefore, this prote-
ase represents a particularly promising enzyme for the develop-
ment of prodrugs for the treatment of cancer and RA. At the
same time, both of these diseases are among the most promis-
ing targets for therapeutic application of CO.[6a]
Figure 2. General design of the protease-triggered CORMs.
The remote positioning of the enzyme-cleavable bond rela-
tively to the planar chiral organometallic moiety would suppress
kinetic resolution during the enzymatic process, thus eliminat-
ing the necessity to control the absolute configuration of the
Herein, we report on the design and synthesis of novel pro- diene-Fe(CO)3 moiety, and conjugation of the enzyme-cleavable
tease-activated CORMs (PT-CORMs) which were expected to ex- moiety through a linker also should facilitate enzymatic hydroly-
hibit improved pharmacological properties (water solubility, sis.[19] Fine-tuning of CO release kinetics is of high importance
Figure 3. Target plasmin-activated CORMs.
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for the development of therapeutically useful CORMs because their mechanism of elimination (1,6-elimination vs. cyclization)
a fast release rate may lead to cytotoxic levels of CO, while a (Scheme 2).[19,20,22] The structure of the dienol complex (Fig-
slow but constant CO release is required to induce a desired ure 2) which is released upon linker self-immolation also has a
biological effect. In the case of PT-CORMs of the general type profound effect on the CO release rate (with the “outer diene”
depicted in Figure 3, the rate of prodrug cleavage and CO re- undergoing oxidative decomposition much faster than the
lease would be determined by superimposition of the rates of “inner diene”).[8b]
the following individual reactions: (1) enzymatic amide bond
hydrolysis; (2) linker self-immolation; (3) “decomposition” of the
dienol-Fe(CO)3 intermediate. The rate of enzymatic cleavage will
depend on the enzyme, the structure of the specifier and prob-
ably also on the linker (as it contributes to the stereochemical
environment around the cleavable amide bond).
Taking all the above considerations together, we selected
four different target structures for this study (Figure 3). The syn-
thesis of PT-CORM 1 (Scheme 3) started with the preparation
of the suitably protected N-acetyl-Gly-d-Ala-l-Phe-l-Lys specifier
containing the self-immolative PAB linker unit using manual
solid phase peptide synthesis.[21] According to a reported pro-
While the tripeptide d-Ala-l-Phe-l-Lys is a well-established cedure commercial 2-chlorotrityl chloride polystyrene resin
specifier for plasmin-activated anti-cancer prodrugs,[15a,18,19b,20] (2-CTC resin) 5 was first treated with thionyl chloride in the
an even more advantageous N-acetyl-Gly-d-Ala-l-Phe-l-Lys se- presence of pyridine followed by addition of the N-Fmoc-pro-
quence was recently reported.[21] As linkers, we selected the tected p-aminobenzyl alcohol to yield the CTC-resin 6.[21] After
well-known p-aminobenzyl (PAB) and 1,2-ethylenediamine- Fmoc-deprotection of 6 under basic conditions the subsequent
carbonyl (EDC) linkers and a combination of p-aminobenzyl and amino acid coupling was carried out using commercially avail-
ethylenediaminecarbonyl fragments (PAB-EDC), which differ in able Fmoc-l-Lys-alloc, Fmoc-l-Phe, Fmoc-d-Ala and Ac-Gly in
Scheme 2. Activation of PT-CORMs with different types of self-immolative linkers.
Scheme 3. Synthesis of PT-CORM 1. Reagents and conditions: (a) SOCl2, pyridine, DCM, 0 °C to r.t., 30 min, then reflux, 4 h; (b) N-Fmoc-p-aminobenzylic
alcohol, pyridine, THF, reflux, 16 h; (c) piperidine, DMF, r.t., 20 min; (d) amino acid, HATU, HOAt, DIPEA, DMF, r.t. (amino acids coupling steps were carried out
with Fmoc-l-Lys-alloc; Fmoc-l-Phe; Fmoc-d-Ala; Ac-Gly sequentially); (e) TFA, DCM, r.t.; (f) SOCl2, THF, r.t., 14 h; (g) rac-9, TBAF, NaH, DMF, 0 °C, 10 min, then r.t.,
14 h; (h) Pd2(dba)3*CHCl3, PPh3, HCOOH, THF, r.t., 14 h. {HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophos-
phate; HOAt = 1-hydroxy-7-azabenzotriazole; DIPEA = diisopropylethylamine; Fmoc = fluorenylmethyloxycarbonyl; alloc = allyloxycarbonyl; TFA = trifluoroacetic
acid}.
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DMF at room temperature.[21] Cleavage of the p-aminobenzyl ref.[8b]) in DMF provided organometallic compound 10 in 70 %
alcohol-peptide conjugate from the resin was carried out by yield. Alloc-deprotection of complex 10 was finally performed
treatment with TFA solution in DCM to provide peptide 7 in with acetic acid in the presence of Pd2(dba)3 as a catalyst to
82 % yield (3 steps).
The benzyl alcohol function in 7 was transformed to the
chloride 8 by nucleophilic substitution using thionyl chloride in
give the desired PT-CORM 1 (as the hydroformate salt) in 93 %
yield.
For the synthesis of the target PT-CORMs 2–4 a set of reac-
THF. Subsequent conjugation of compound 8 with the cyclo- tive cyclohexadiene-Fe(CO)3 complexes, i.e. 11 and rac-12 with
hexadiene-Fe(CO)3 complex rac-9 (prepared according to an RO function at the “outer” position of the cyclohexadiene-
Scheme 4. Synthesis of cyclohexadiene-Fe(CO)3 complexes. Reagents and conditions: (a) TBAF, THF, 0 °C, 10 min, then 4-nitrophenyl chloroformate, DIPEA,
1.5 h (TBAF = tetra-n-butyl ammonium fluoride).
Scheme 5. Synthesis of PT-CORMs 2 and 3. Reagents and conditions: (a) SOCl2, pyridine, DCM, 0 °C to r.t., 30 min, then reflux, 4 h; (b) Fmoc-Lys(Alloc)-OH,
DIPEA, DCM, r.t., 14 h; (c) piperidine, DMF, r.t., 20 min; (d) amino acid, HATU, HOAt, DIPEA, DMF, r.t. (amino acid coupling steps were carried out with Fmoc-l-
Phe; Fmoc-d-Ala; Ac-Gly sequentially); (e) TFA, DCM, r.t.; (f) N-Boc-N-Me-ethylenediamine 17, Py-BOP, HOBt, DIPEA, DMF, r.t., overnight; (g) HCl aq, EtOAc, r.t.,
2 h; (h) 11 + rac-12, DIPEA, DMF, r.t., 1 h; (i) rac-14, DIPEA, DMF, 0 °C, 30 min; (j) Pd2(dba)3*CHCl3, PPh3, HCOOH, THF, r.t., 14 h. {Boc = tert-butyloxycarbonyl;
Py-BOP = (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; HOBt = 1-hydroxybenzotriazole}.
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Scheme 6. Synthesis of PT-CORM 4. Reagents and conditions: (a) 4-nitrophenyl chloroformate, pyridine, THF; (b) N-Boc-N,N′-dimethyl-1,2-ethylenediamine 23,
DMF, –20 °C, 2 h; (c) HCl aq, EtOAc, r.t., 14 h; (d) rac-12, Et3N, DMF, r.t., 10 min; (e) Pd2(dba)3*CHCl3, PPh3, HCOOH, THF, r.t., overnight.
Fe(CO)3 moiety and rac-14 with the “inner” position of RO a literature protocol[23] and attached to peptide 16 using PyBOP
group, were prepared according to Scheme 4. Following our as a coupling reagent to afford 18 in 98 % yield. Subsequent
reliable protocols for the selective synthesis of acyloxy-substi- Boc-deprotection gave the peptide 19 which was smoothly
tuted cyclohexadiene-Fe(CO)3 complexes in both regioisomeric converted into the PT-CORM precursors 20 and 21 by reaction
series (as racemates) we first prepared the TIPS-protected com- with complexes 11/rac-12 and rac-14, respectively, in the pres-
plexes as storable intermediates.[8b] The isomeric TIPS-protected ence of DIPEA in DMF. The separation of 11 and rac-12 was not
dienol complexes rac-9[8b] and rac-13[8b] were then reacted necessary as both compounds reacted with 19 to give the same
with 4-nitrophenyl chloroformate in the presence of TBAF and product. Finally, Pd-catalyzed alloc-deprotection afforded the
DIPEA in THF to give the compounds 11, rac-12 and rac-14 target PT-CORMs 2 and 3 (as hydroformate salts) in an isolated
(Scheme 4).
yield of 75 and 90 %, respectively (Scheme 5).
PT-CORMs 2 and 3 which contain EDC linker were prepared
For the preparation of PT-CORM 4 alcohol 7 was activated
according to Scheme 5. After pre-activating the 2-CTC-resin 5 using 4-nitrophenyl chloroformate in the presence of pyridine
with SOCl2 as before (Scheme 3) reaction with Fmoc-Lys(alloc)- to give carbamate 22 in 82 % yield (Scheme 6).[21] N-Boc-N,N′-
OH in the presence of DIPEA in DCM afforded the functional- dimethyl-1,2-ethylenediamine 23[24] was attached to com-
ized resin 15 which was further converted to peptide 16 by pound 22 in DMF yielding 24, which was subjected to Boc de-
successive coupling with the amino acids Fmoc-l-Phe, Fmoc-d- protection under acidic conditions leading to amine 25 in 23 %
Ala and Ac-Gly according to Scheme 5 (81 % over three steps). yield over two steps. Complex 26 was formed by coupling of
the conjugate 25 with the organometallic unit rac-12 in the
presence of Et3N in DMF in 85 % yield. After alloc-deprotection
the desired target complex 4 was isolated in 80 % yield, again
as the hydroformate salt.
The linker N-Boc-N-methyl-ethylenediamine 17 was prepared
following a literature protocol[23] and attached to peptide 16
using PyBOP as a coupling reagent to afford 18 in 98 % yield.
Subsequent Boc-deprotection gave the peptide 19 which was
smoothly converted into the PT-CORM precursors 20 and 21 by
reaction with complexes 11/rac-12 and rac-14, respectively, in
the presence of DIPEA in DMF. The separation of 11 and rac-12
was not necessary as both compounds reacted with 19 to give
Conclusions
the same product. Finally, Pd-catalyzed alloc-deprotection af- We designed a new chemical architecture for potential tissue-
forded the target PT-CORMs 2 and 3 (as hydroformate salts) in selective protease-activated CO-releasing molecules (PT-
an isolated yield of 75 and 90 %, respectively (Scheme 5). The CORMs) based on our previous experience with acyloxy-cyclo-
linker N-Boc-N-Me-ethylenediamine 17 was prepared following hexadiene-Fe(CO)3 complexes as enzyme-triggered CORMs. For
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23.8, 22.5, 17.4. HRMS (ESI): calcd. for C38H47FeN6O9 [M – HCOO]+
this purpose we connected a plasmin-specifying peptide se-
quence to the organometallic CORM unit through a self-immo-
lative linker (p-aminobenzyl for PT-CORM 1), 1,2-ethylenedi-
aminecarbonyl for PT-CORMs 2 and 3, and a combined linker
for PT-CORM 4. These challenging and non-trivial target struc-
tures were then synthesized in a straightforward manner ex-
ploiting a combination of solid-phase peptide synthesis and so-
lution chemical synthesis. Late-stage attachment of the (race-
mic) organometallic unit and Pd-catalyzed cleavage of an alloc-
protected lysine side chain afforded the target compounds in
good yield, which all were isolated as their hydroformate salts
in high purity and proved to be stable under ambient condi-
tions.
While the synthesized compounds are currently under bio-
logical evaluation, we are confident that the methodology for
their synthesis, which we have disclosed here, provides a valu-
able basis for the synthesis of the development also of other
protease-activated CORM prodrugs with tunable pharmacologi-
cal properties in the future.
787.2748, found 787.2749. IR (ATR): ν = 2037 (s, Fe(CO)3), 1952 (s,
˜
Fe(CO)3) cm–1
.
Synthesis of the Acyloxydiene-Fe(CO) Building Blocks 11/rac-
12: A solution of rac-9 (1 equiv., 5 mmol) in anhydrous THF (50 mL)
was cooled down to 0 °C and TBAF (1
M in THF, 1.05 equiv.,
5.25 mmol, 5.25 mL) was added dropwise. The mixture was stirred
for 10 min before DIPEA (4 equiv., 20 mmol, 3.39 mL) and 4-nitro-
phenyl chloroformate (4 equiv., 20 mmol, 4.04 g) were sequentially
added. After stirring the suspension for 1.5 h at 0 °C the solvent was
removed under reduced pressure and the residue was subjected
to column chromatography on silica gel (Cy/EtOAc, 5:1) to yield
compound 11 (550 mg, 1.11 mmol, 22 %) as a yellowish solid and
compound rac-12 (750 mg, 1.87 mmol, 37 %) as a brown solid.
Analytical data for 11: 1H NMR (300 MHz, CDCl3): δ = 5.67 (ψ s,
2H), 3.51 (ψ s, 2H), 3.02–2.76 (m, 2H), 1.96–1.67 (m, 4H), 1.70–1.38
(m, 4H). 13C NMR (75 MHz, CDCl3): δ = 210.3, 129.2, 79.5, 57.9, 52.0,
25.0, 23.5. HRMS (ESI): calcd. for C19H15O9Fe2 [M + H]+ 498.9409,
found 498.9413; calcd. for C19H14O9Fe2Na [M + Na]+ 520.9229,
found 520.9232. IR (ATR): ν = 2042 (s, Fe(CO)3), 1950 (s, Fe(CO)3),
˜
1772 (C=O) cm–1. Analytical data for rac-12: 1H NMR (300 MHz,
3
3
CDCl3): δ = 8.31 (d, J = 9.1 Hz, 2H), 7.45 (d, J = 9.1 Hz, 2H), 5.72
3
4
3
4
(dd, J = 6.5 Hz, J = 2.5 Hz, 1H), 3.55 (ψ q, J = 2.5 Hz, J = 2.5 Hz,
Experimental Section
Synthesis of Complex 1: The TIPS-protected complex rac-9
(2 equiv., 0.9 mmol, 353 mg) was dissolved in anhydrous DMF
(4.5 mL) under argon atmosphere and the resulting solution was
3
3
1H), 2.92 (dt, J = 6.5 Hz, J = 2.5 Hz, 1H), 1.96–1.69 (m, 2H), 1.68–
1.36 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 210.2, 155.2, 151.4,
145.9, 129.2, 125.6, 121.8, 79.6, 57.9, 57.9, 24.9, 23.51. MS (EI, 70 eV):
m/z (%) = 473 ([M – CO]+), 345 ([M – 2CO]+), 317 ([M – 3CO]+,
100 %), 285, 261 ([M – Fe(CO)3]+). IR (ATR): ν = 2060 (s, Fe(CO)3),
˜
cooled to 0 °C. TBAF (1
M solution in THF, 2.2 equiv., 0.986 mmol,
1969 (s, Fe(CO)3), 1762 (C=O), 1520 and 1348 (m, NO2) cm–1
.
986 μL) and sodium hydride (60 wt.-% dispersion in mineral oil,
2.2 equiv., 0.986 mmol, 39 mg) were sequentially added and the
reaction mixture was stirred for 10 min at 0 °C. Then, a solution
of peptide 8 (1 equiv., 0.448 mmol, 300 mg) in anhydrous DMF
(4.5 mL) was added, the ice bath was removed and the mixture was
stirred overnight. After removing the solvents in vacuo the product
was purified by flash chromatography (SiO2, DCM/MeOH, 15:1) to
give crude 10 (275 mg, 70 %) as a pale brownish solid. A flame-
dried Schlenk flask was charged with Pd2(dba)3*CHCl3 (0.1 equiv.,
Synthesis of Complex 2: Peptide 19 (1 equiv., 0.61 mmol, 390 mg)
was dissolved in anhydrous DMF (6 mL) under argon. Then a solu-
tion of a mixture of 11 (0.45 equiv., 0.273 mmol, 136 mg) and rac-
12 (0.86 equiv., 210 mg, 0.524 mmol) in anhydrous DMF (6 mL) was
added followed by DIPEA (10 equiv., 6.1 mmol, 1.06 mL). The result-
ing dark orange solution was stirred for 1 h at room temperature.
Then DMF was removed under reduced pressure (vacuum line, bath
temperature 45–50 °C) and the residue was subjected to column
0.020 mmol, 20 mg) and PPh3 (0.4 equiv., 0.078 mmol, 20 mg) be- chromatography on silica gel (DCM/MeOH, 10:1) to yield 20
fore anhydrous THF (2 mL) and HCOOH (10 equiv., 1.95 mmol,
73.5 μL) were sequentially added. The mixture was stirred for 10
minutes at room temperature before a suspension of 10 (1 equiv.,
0.195 mmol, 170 mg) in anhydrous THF (8 mL) was transferred to
the stirred mixture in the Schlenk flask containing the Pd-catalyst.
(350 mg, 66 %, as a 1:1 mixture of epimers) as a white solid. A
flame-dried Schlenk flask was charged under argon with
Pd2(dba)3*CHCl3 (0.1 equiv., 0.05 mmol, 52 mg) and PPh3 (0.4 equiv.,
0.2 mmol, 52 mg) before anhydrous THF (5 mL) and HCOOH
(10 equiv., 5 mmol, 189 μL) were sequentially added, and the mix-
The resulting orange-yellow suspension was stirred overnight to ture was stirred for 10 minutes at room temperature. Then, a sus-
form a yellowish gel. The latter was diluted with THF (10 mL) and
the precipitate was filtered off using glass frit and washed with THF
and Et2O. The precipitate was left on the air for ca. 30 minutes
before the product was washed off the glass frit with MeCN/MeOH
pension of 20 (1 equiv., 0.5 mmol, 433 mg) in anhydrous THF
(15 mL) was transferred to the stirred mixture in the Schlenk flask
containing the Pd-catalyst. The resulting orange-yellow suspension
was stirred overnight to form a brown gel-like mixture. The latter
(1:1 v/v, 50 mL). The solvents were removed under reduced pressure was diluted with MeOH (10 mL), and after addition of silica gel
to give complex 1 (149 mg, 93 %) as a pale yellow solid which did
the solvents were evaporated to yield the crude product directly
absorbed on silica gel. The product was finally isolated using
not show any impurities in NMR. 1H NMR (600 MHz, CD3OD):
3
3
δ = 8.56 (s, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), column chromatography on silica gel (EtOAc/MeCN/MeOH/HOH,
7.29–7.14 (m, 5H), 5.39 (dd, 3J = 6.7 Hz, 4J = 2.2 Hz, 1H), 4.96 (d,
2J = 11.1 Hz, 1H), 4.70 (d, 2J = 11.1 Hz, 1H), 4.54 (dd, 3J = 9.9 Hz,
3:1:1:1 + 0.05 vol.-% HCOOH). The solvents were removed under
reduced pressure using MeCN for azeotropic removal of water. The
resulting solid was dissolved in HPLC-grade MeOH/MeCN (1:1 v/v)
3J = 4.9 Hz, 1H), 4.48 (dd, J = 9.5 Hz, J = 4.8 Hz, 1H), 4.21 (q, J =
3
3
3
7.1 Hz, 1H), 3.80 (d, 2J = 16.5 Hz, 1H), 3.71 (d, 2J = 16.5 Hz, 1H), 3.51 and the solution was filtered through a syringe filter (0.2 μm, PTFE).
3
4
2
3
(dd, J = 5.3 Hz, J = 2.2 Hz, 1H), 3.26 (dd, J = 14.1 Hz, J = 4.9 Hz,
After removal of the solvents and drying under reduced pressure
for 8 h product 2 (302 mg, 73 %) was obtained as a pale yellowish
1H), 2.99 (dd, 2J = 14.1 Hz, 3J = 9.9 Hz, 1H), 2.96–2.89 (m, 2H), 2.87–
2.82 (m, 1H), 2.01–1.91 (m, 1H), 1.95 (s, 3H), 1.90–1.80 (m, 1H), 1.77– solid (mixture of epimers at the planar chiral unit). 1H NMR
1.64 (m, 4H, 24-H), 1.62–1.40 (m, 4H, 23-H), 1.20 (d, 3J = 7.1 Hz, 3H).
(500 MHz, CD3OD): δ = 8.48 (s, 1H), 7.80–7.67 (m, 4H), 7.35–7.16 (m,
13C NMR (126 MHz, CD3OD): δ = 212.9, 175.7, 173.9, 173.7, 172.1, 1H), 5.77–5.70 (m, ≈ 0.5H), 5.70–5.60 (m, ≈ 0.5H), 4.56–4.48 (m, 1H),
171.8, 140.4, 139.4, 138.5, 133.6, 130.2, 129.8, 129.5, 127.8, 121.4, 4.35–4.28 (m, 1H), 4.22–4.13 (m, 1H), 3.82 (s, 2H), 3.57–3.22 (m, 6H,
70.4, 70.2, 56.7, 55.1, 52.7, 51.0, 43.5, 40.6, 37.8, 32.1, 28.2, 25.8, 24.5,
12-Ha), 3.02–2.84 (m, 7H), 2.00 (s, 3H), 1.94–1.36 (m, 10H), 1.21–1.16
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Communication
(m, 3H). 13C NMR: Multiple signals sets are observed due to the
presence of diastereomers and amide rotamers; see the SI. HRMS
(ESI): calcd. for C35H48N7O10Fe [M – HCOO]+ 782.2807, found
782.2817; calcd. for C35H47N7O10FeNa [M – HCOOH + Na]+ 804.2626,
of diastereomers and amide rotamers; see the SI. HRMS (ESI):
calcd. for C44H57N8O12Fe [M – HCOO]+ 945.3440, found 945.3446;
calcd. for C44H56N8O12FeNa [M – HCOOH + Na]+ 967.3259, found
967.3252. IR (ATR): ν = 3268 (s br, NH), 2043 (s, Fe(CO)3), 1961 (s,
˜
found 804.2628.). IR (ATR): ν = 3269 (s br, NH), 2044 (s, Fe(CO)3),
Fe(CO)3), 1724 and 1698 (m, C=O), 1635 and 1603 (s, C=O), 1544
˜
1961 (s, Fe(CO)3), 1635 and 1612 (s, C=O), 1548 (s, C=O) cm–1
.
(s, C=O) cm–1
.
Supporting Information (see footnote on the first page of this
article): For further experimental details including NMR signal as-
signments, see the supporting information.
Synthesis of the Acyloxydiene-Fe(CO) Building Block rac-14: A
solution of rac-13 (1 equiv., 1 mmol) in anhydrous THF (10 mL) was
cooled to 0 °C and TBAF (1
M in THF, 1.05 equiv., 1.05 mmol,
1.05 mL) was added dropwise. After 10 min, DIPEA (4 equiv.,
4 mmol, 0.68 mL) and 4-nitrophenyl chloroformate (4 equiv.,
4 mmol, 0.81 g) were sequentially added and the resulting suspen-
sion was stirred for 1.5 h at 0 °C. After removal of the solvent under
reduced pressure the residue was subjected to column chromatog-
raphy on silica gel (Cy/EtOAc, 5:1) to yield rac-14 (335 mg, 84 %) as
a brownish solid. 1H NMR (300 MHz, CDCl3): δ = 8.28 (d, 3J = 9.1 Hz,
2H), 7.37 (d, 3J = 9.1 Hz, 2H), 5.55 (d, 3J = 4.4 Hz, 1H), 5.16 (ψ t, 3J =
Acknowledgments
This work was supported by the Alexander von Humboldt foun-
dation (postdoctoral fellowship to N. S.), the Deutsche For-
schungsgemeinschaft (Project SCHM857/20-1) and the Russian
Foundation for Basic Research (Project №18-503-12087).
3
5.1 Hz, 1H), 3.18 (dt, 3J = 5.1 Hz, J = 2.0 Hz, 1H), 2.43–2.19 (m, 1H),
2.04–1.62 (m, 3H, 6-H). 13C NMR (75 MHz, CDCl3): δ = 210.8, 155.4,
150.8, 145.7, 125.5, 122.0, 105.0, 81.3, 79.7, 61.1, 26.4, 24.0. IR (ATR):
Keywords: CO releasing molecules · Peptides · Iron
complexes · Enzymes · Protecting groups
ν = 2047 (s, Fe(CO)3), 1970 (s, Fe(CO)3), 1764 (C=O), 1530 and 1347
˜
(m, NO2), 1207 (s, C-O) cm–1
.
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Synthesis of Complex 3: Peptide 19 (1 equiv., 0.61 mmol, 320 mg)
and rac-14 (1.2 equiv., 0.574 mmol, 230 mg) were dissolved in anhy-
drous DMF (10 mL) under argon and the solution was cooled to
0 °C. Then, trimethylamine (5 equiv., 2.39 mmol, 333 μL) was added
and the mixture allowed to stir for 30 min before the solvent was
removed under reduced pressure. Purification by chromatography
on silica gel (DCM/MeOH, 10:1) yielded 21 (320 mg, 77 %) as a
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scribed above in the synthesis of complex 2 0.404 mmol of 21 were
reacted to give 3 (250 mg, 90 %) as a colorless solid. 1H NMR
(400 MHz, [D6]DMSO): δ = 8.61–7.90 (m, 6H, NH), 7.33–7.09 (m, 5H),
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3
5.65 (d, J = 4.3 Hz, 1H), 5.36 (ψ s, 1H), 4.47 (m, 1H), 4.30–4.06 (m,
1H), 3.66 (m, 2H), 3.35–3.14 (m, 4H), 3.09 (dd, 2J = 13.9 Hz, 3J =
3.9 Hz, 1H), 2.97–2.62 (m, 5H), 2.21–2.00 (m, 1H), 1.93–1.71 (m, 4H),
1.71–1.16 (m, 8H), 0.98 (d, 3J = 6.9 Hz, 3H). 13C NMR: Multiple signal
sets are observed due to the presence of diastereomers and amide
rotamers; see the SI. HRMS (ESI): calcd. for C35H48N7O10Fe [M –
HCOO]+ 782.2807, found 782.2809; calcd. for C35H47N7O10FeNa [M
– HCOOH + Na]+ 804.2626, found 804.2620. IR (ATR): ν = 3268 (s br,
˜
NH), 2044 (s, Fe(CO)3), 1962 (s, Fe(CO)3), 1662 and 1635 and 1602
(s, C=O), 1548 (s, C=O) cm–1
.
Synthesis of Complex 4: To a solution of peptide 25 (1 equiv.,
0.074 mmol, 60 mg) in anhydrous DMF (1.6 mL) stirred under argon
at room temperature were sequentially added rac-12 (1.5 equiv.,
0.111 mmol, 45 mg) and trimethylamine (2 equiv., 0.148 mmol,
21 μL). After 10 minutes the solvent was removed under reduced
pressure and the resulting solid was purified by column chromatog-
raphy on silica gel (DCM/MeOH, 10:1) to give 26 (71 mg of still
containing traces of DMF and p-nitrophenol; i.e. 64.5 mg, 85 %)
as a white solid which was further used following the same alloc
deprotection protocol as described above in the synthesis of com-
plex 2. 0.058 mmol of 26 were reacted to give 4 (46 mg, 80 %) as
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a pale brownish solid. H NMR (500 MHz, CD3OD): δ = 8.52 (s, 1H),
3
3
7.63 (d, J = 7.7 Hz, 2H), 7.36 (d, J = 7.7 Hz, 2H), 7.29–7.14 (m, 5H),
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(m, 10H), 2.01–1.62 (m, 7H), 1.58–1.37 (m, 6H), 1.20 (d, J = 7.0 Hz,
1H). 13C NMR: Multiple signal sets are observed due to the presence
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Received: August 16, 2019
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Communication
Peptide Conjugates
Potentially tissue-selective protease-
activated CO-releasing molecules (PT-
CORMs) were designed and synthe-
sized by conjugating a protease-speci-
fying peptide to an oxy-cyclohexadi-
ene-Fe(CO)3 unit through a self-immo-
lative linker.
N. S. Sitnikov, Y. B. Malysheva,
A. Y. Fedorov,* H.-G. Schmalz* ....... 1–9
Design and Synthesis of New Prote-
ase-Triggered CO-Releasing Pept-
ide–Metal-Complex Conjugates
DOI: 10.1002/ejoc.201901206
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