Minimalistic peptidic scaffolds harbouring an artificial carbene-containing amino acid modulate reductase activity

Article abstract of DOI:10.1039/d1cc03158a

Inspired by the boom of new artificial metalloenzymes, we developed an Fmoc-protected histidinium salt (Hum) as N-heterocyclic carbene precursor. Hum was placedviasolid-phase peptide synthesis into short 7-mer peptides. Upon iridation, the metallo-peptidic construct displayed activity in catalytic hydrogenation that outperforms small molecule analogues and which is dependent on the peptide sequence, a typical feature of metalloenzymes.

Full text of DOI:10.1039/d1cc03158a

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DOI: 10.1039/D1CC03158A
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Minimalistic peptidic scaffolds harbouring an artificial carbene-
containing amino acid modulate reductase activity
Karst Lenzen,#a Matteo Planchestainer,#a Isabelle Feller,a David Roura Padrosa,a Francesca
Received 00th January 20xx,
Accepted 00th January 20xx
Paradisi *a and Martin Albrecht *a
DOI: 10.1039/x0xx00000x
Inspired by the boom of new artificial metalloenzymes, we precursors in amino acids, we got intrigued to broaden the
developed an Fmoc-protected histidinium salt (Hum) as N- scope by incorporating a carbene precursor into peptide
heterocyclic carbene precursor. Hum was placed via solid-phase sequences and test the effect of different amino acid scaffolds
peptide synthesis into short 7-mer peptides. Upon iridation, the in catalysis. Therefore, the histidinium salt Fmoc-Hum-OH
metallo-peptidic construct displayed activity in catalytic (Scheme 1, abbreviated one letter code Ḧ) was designed with a
hydrogenation that outperforms small molecule analogues and Fmoc protection group which allows for use in standard solid-
which is dependent on the peptide sequence, a typical feature of phase peptide synthesis (SPPS). Here, we show a simple series
metalloenzymes.
of palindromic heptapeptides with which we probe the
effectiveness of coupling Hum in SPPS. Iridium was chosen as a
metal center for binding the carbene, as Ir–NHC complexes have
shown high activity in a range of catalytic applications,12
moreover, the Ir–CNHC bond is exceptionally stable under acidic
and basic conditions.13 This approach furnished metallo-
peptides with pseudo reductase activity, demonstrated in the
hydrogenation of acetophenone as model reaction.
The artificial amino acid Hum as NHC precursor was prepared
by methylation of Boc-His-OH, which yielded Boc-Hum-OMe in
good yields (Scheme 1).14 Sequential protecting group
modifications gave Fmoc-Hum-OH over 4 steps in 47% overall
yield. A direct Boc/Fmoc exchange after ester hydrolysis via the
unprotected Hum gave only traces of the desired product in a
complex mixture. Fmoc-Hum-OH was analyzed by NMR
spectroscopy, MS, and its purity confirmed by microanalysis.
Diagnostic NMR signals include the two singlets due to
chemically distinct N-CH3 groups (dH 3.51, 3.48) and two
aromatic resonances at dH 6.87 and 8.31 for Hd and He,
respectively. Derivatization of Fmoc-Hum-OH via Hum–OMe
with (S)-Mosher’s acid indicated at least 95% retention of
enantiopurity (ESI) and negligible racemization during the Boc
deprotection step of the phenacyl (PAc) protected ester
group.15
The implementation of abiotic reactivity is considered to be one
of the most powerful strategies to broaden the toolbox of
biocatalysis.1 Non-natural transformations have been evoked,
for example, by directed evolution of natural metalloenzymes,
e.g. silylation with P450,2 by site directed mutagenesis using
(non-)natural amino acids,3 or by the incorporation of a
synthetic co-factor with a specific abiotic transition metal
center.4 This latter approach also allowed for incorporating N-
heterocyclic carbene (NHC) metal complexes into a protein
scaffold to produce evolvable artificial metalloenzymes that
catalyze olefin metathesis5 and hydrogenation.6
The use of N-heterocyclic carbene complexes to generate
minimalistic peptidic scaffolds displaying pseudo-enzymatic
activity: on one handside, NHCs have been standing out as
facilitator ligands in homogeneous catalysis7 and, on the other,
the side chain of histidine (His) provides the imidazole skeleton
of NHCs and may therefore potentially bind a metal center via
classical N- or carbenic C-coordination.8 Previous work in our
group9 and others10 has shown that histidylidene complexes are
accessible, though incorporation into oligopeptides required
the introduction of an external NHC ligand.11
Considering the potential existence of carbenes in biological
systems,2,3,8 and the possibility to incorporate carbene-
Cl–
N+
I–
N+
N
N
N
NH
OH
i)
ii)–v)
OH
a. Department of Chemistry, Biochemistry & Pharmaceutical Sciences, University of
Bern, Freiestrasse 3, 3012 Bern, Switzerland; E-mail:
Francesca.paradisi@dcb.unibe.ch, martin.albrecht@dcb.unibe.ch.
# These authors contributed equally
O
BocHN
FmocHN
BocHN
O
O
O
Boc-His-OH
Boc-Hum-OMe
Fmoc-Hum-OH
† Electronic Supplementary Information (ESI) available: Synthetic and catalytic
procedures, conformation movie, and analytical data. See DOI: 10.1039/x0xx00000x
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Scheme 1. Synthesis of Fmoc-protected Hum (Ḧ). Reagents and
conditions: i) MeI, MeCN, 40 C; ii) LiOH, MeOH/H2O, rt; iii) PAc-Br,
KF, DMF, rt; iv) HCl, dioxane, then immediately FmocCl, Na2CO3,
MeCN, rt; v) Zn, HOAc, then HCl, dioxane, rt.
mmol/g pre-loading). Identity and purity were analyzed by high-
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resolution ESI-MS, LC-MS and NMR sDpOeIc:t1r0o.1s0c3o9p/Dy1C(C†0).315F8oAr
example, for the sequence ASAḦASA with two Ser and Hum and
alternating Ala, 1H–1H COSY identified the Hum CαH-proton as a
distinct triplet at δH 4.61, which is well-separated from the CαH-
protons of the other amino acids (δH 4.18–4.35). The Hum Hd
and He appeared as singlets at δH 7.17 and 8.52 ppm,
Fmoc-Hum-OH has the appropriate functionalization for
application in SPPS and has been used to prepare a set of
palindromic heptapeptides AXAḦAXA with varying amino acids
X in the 2- and 6-position and Hum (Ḧ) in the central position
(Scheme 2). To test the compatibility of the Hum towards
coordination of metals in the presence of different functional
groups, X was permuted with all 20 natural amino acids. This
afforded 20 heptapeptides that were unfunctionalized neutral
(X = Gly), hydrophobic (X = Ala, Val, Pro, Ile, Leu, Met), aromatic
(X = Phe, Tyr, Trp), positively charged (X = His, Arg, Lys),
negatively charged (X = Asp, Glu) or polar uncharged (X = Ser,
Asn, Tyr, Gln, Cys). While coupling of the first three amino acids
on the Wang resin followed standard procedures,16 coupling of
Fmoc-Hum-OH required longer reaction times (5 vs 1-2 h) to
reach completion (TNBS control). Also, coupling of the
subsequent Ala was performed twice to reach optimal results.
Subsequent introduction of X and A proceeded again according
to standard protocols.
1
respectively. The presence of just one set of signals in the H
and 13C NMR spectra indicate the presence of just one isomer,
suggesting that either the D-Hum epimer is not distinguishable
by NMR analysis (cf Mosher acid analysis) or that only the L-
enantiomer is incorporated into the peptide.
Installation of the catalytic iridium center was accomplished on
the resin-bound heptapeptide by treatment with Ag2O to form
the putative carbene silver intermediate, followed by
transmetalation with [IrCl2Cp*]2.‡ After incubation for 24 h, the
metallopeptide was cleaved from the resin with TFA, a
procedure that is applicable because the Ir–CNHC bond is
remarkably stable under acidic conditions. While this procedure
was successful for most palindromic sequences, those with X =
Val, Trp, or His gave lower yields, the latter presumably because
of interference of the heteroaromatic side chain with the metal
center. Moreover, the procedure failed when Cys or Met were
used as amino acids, which was attributed to the strong affinity
of Ag to sulfur ligands and therefore a preferred reactivity of
Ag2O with these side chains rather than the Hum.
O
FmocHN
Fmoc-Ala-Wang resin
Analysis by LC-MS of crude samples after resin cleavage
revealed only partial metalation, which was attributed to partial
hydrolytic Ir–C bond scission in the resin cleavage step due to
the presence of TFA rather than to an incomplete metalation.
Revers phase HPLC purification yielded the pure peptidic
scaffolds AXAḦAXA–Ir (LC-MS). Analysis by HR-MS revealed a
most significant signal for [AXAḦAXA–Ir –2Cl]2+ for all peptides
except for the Lys-containing heptamer, for which z = 3 due to
the basic side chain (†). The NMR spectra of these systems were
consistently very broad, though they clearly revealed the Cp*
proton signals around 1.6 ppm in the correct integral ratio.
Depending on the amino acid sequence, this signal is split in two
or more different signals, which however coalesce to a single
resonance at 50 °C. Therefore, the different Cp* signals were
attributed to distinct rotamers that show restricted
interconversion due to the metal fragment bound to Hum,
rather than epimers.
Since NHC iridium complexes are established hydrogenation
catalysts, we investigated the activity of this minimalistic
peptidic scaffolds in catalytic hydrogenation in citrate buffer at
pH 3. All AXAḦAXA–Ir constructs showed activity in the
hydrogenation of acetophenone as model substrate, albeit
without any enantiomeric excess (ee <3%). Reminiscent to
metalloenzymes, the activity of the heptapeptide shows
considerable dependence on the type of amino acids in close
proximity to the active site (Table 1, entries 1–18). For example,
there is an order of magnitude difference between X = R (TOF =
3 h–1) and X = Y (TOF = 30 h–1; entry 11 vs 8).§ Some general
trends from this initial activity screen can be deduced. For
example, aromatic side chains (X = F, Y) have a positive effect on
the catalytic activity, possibly because of suitable interactions
[i), ii)]6
i), iii)
Cl–
N
N+
O
R
O
O
R
O
H
N
H
N
H
H
N
N
N
H
N
N
H
H
O
O
O
O
resin-AXAḦAXA
iv)–vi)
Ir
Cl
Cl
N
O
N
O
R
O
O
R
O
H
N
H
N
H
H
N
N
N
H
N
N
H
OH
H
O
O
O
AXAḦAXA–Ir
Scheme 2. Solid-phase peptide synthesis (SPPS) of the palindromic
AXAḦAXA heptapeptide and iridation to form a mini-
metalloenzyme with a NHC iridium active site. Reagents and
conditions: i) piperidine in DMF; ii) Fmoc-Aaa-OH, oxyma, collidine,
DIC in NMP; iii) AcOAc in CH2Cl2,; iv) Ag2O, Me4NCl in CH2Cl2/MeCN;
v) [IrCpCl2]2 in CH2Cl2/MeCN; vi) TFA in H2O. X = permutation of all
20 natural amino acids.
TFA-mediated cleavage of the heptapeptide followed by revers-
phase HPLC purification yielded the apo-peptides sub-gram
quantities (about 20 mg per 50 mg Wang resin with a 0.4–0.8
2 | J. Name., 2012, 00, 1-3
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with the substrate. Potentially coordinating side chains reduce
the activity (X = H, K) or induce an induction period (X = E, but
not D, probably because the additional CH2 group in E enables
metal binding). Bulkiness of the side chain has some inhibiting
effect (S vs T, N vs Q), yet this effect was not observed in
hydrophobic side chains. These variations indicate that these
mini scaffolds can be tailored despite their small size. Moreover,
the oligopeptide backbone is highly relevant for the catalytic
activity as the iridium-complex 1 bound to protected Hum or a
simple NHC iridium analogue (2) showed much lower activity
and reached a modest <20% conversion after 24 h (cf >90%
conversion with selected mini scaffolds with X = A, F, G, S, Y)
under identical reaction conditions (entries 20,21, Fig. S1). The
metal precursor [IrCl2Cp*]2 does not show any detectable
activity (entry 19).
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DOI: 10.1039/D1CC03158A
Ir
Ir
Cl
Cl
Cl
Cl
N
O
N
N
N
OMe
BocHN
1
2
Chart 1. Schematic of small molecule catalysts
1 and 2.
To shed some light on the structural implications imparted by
the variations in the amino acid sequence, molecular dynamics
simulations of all 18 catalytically active heptapeptides were
performed (†). The peptides were modelled initially with His
rather than the Hum–Ir in central position. The Hum–Ir was then
constructed as a constrained entity based on reported crystal
structure data using the UCSF Chimera software. The
conformational changes of the metal-free heptapeptides in 20
ns intervals indicate that despite their short size, most
heptapeptides adopt stable secondary structures (Figure S2–S4,
see also CD spectra in Figure S5).17 While neither the root mean
square deviation of the conformational changes of the
heptapeptide nor the number of clusters from the simulations
correlate with the catalytic activity, the histidine exposure
revealed a positive correlation with the TOFmax (Figures S6,
Table S1). For example, AYAHAYA with two Tyr adopts
secondary structures in which the central His is well-exposed for
solvent and substrate accessibility. Closer inspection of the
structures indicates a conformationally stable interaction
between one phenol side chain and the central His unit, also
when bound to Ir in the AYAḦAYA-Ir variant (Fig. 1). Similar
His/Tyr interactions have been noted previously.18 The
correlation between His exposure and catalytic activity for
subsets of the heptapeptides (Figure S6) constitutes a key
concept of metalloenzyme engineering and offers an attractive
approach for further optimization of the catalytic performance,
especially upon increasing the length of the peptide sequence.
Table 1. Catalytic hydrogenation of acetophenone.a
OH
OH
O
cat. AXAḦAXA–Ir
+
H2 (1 atm)
Citrate b./tBuOH (4:1, pH 3)
40 oC
entry category
X b
TOFmax (h-1)c conversion (%)d
1
2
3
4
5
6
7
8
unfunctionalized
G
A
V
P
I
L
F
Y
W
H
R
K
D
E
S
N
20
17
18
18
16
13
24
30
12
n.d.
3
95
91
86
77
83
82
94
96
60
<2
40
81
80
78
94
88
82
70
<2
18
19
hydrophobic
aromatic
9
10
11
12
13
14
15
16
17
18
19
20
21
+ charge
– charge
9
17
17
17
19
7
10
n.d.
2
polar uncharged
reference
T
Q
[IrCl2Cp*]2
1
2
1
a
Figure 1. Superimposition of most stable conformations deduced
from molecular dynamics simulation of representative
heptapeptides AXAHAXA containing a central His and X = Tyr, Ser,
Gly, Val, and Arg) from two different angles (a and b). The phenol
residue of Tyr remains in close proximity of the His (see ESI for time-
deconvoluted movies of conformational changes for two
metallopeptides AXAḦAXA–Ir with X = Tyr and Lys).
General reaction conditions: acetophenone (10 µmol), [Ir] (0.1 µmol,
1 mol%), citrate buffer pH 3 / tBuOH (1 mL, 4:1 v/v), 40 oC, H2
atmosphere; b X = M, C not evaluated since metalation did not proceed;
c TOFmax determined from the rate at the steepest section for each entry,
n.d. not determined; d conversion determined by GC, all ee <3%.
In summary, a versatile pre-carbene amino acid was developed,
and was incorporated into oligopeptides to generate
minimalistic protein-like assemblies containing a metal-carbene
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Chem. Rev., 2018, 118, 142–231; H. M. Key, P. Dydio, D. S.
active site. The introduction of an Fmoc-protecting group on the
N-terminus of the amino acid allowed for the use of SPPS as
synthetic approach for de novo peptide synthesis. Metalation
was performed successfully with an Ir-precursor on the resin-
bound peptides, followed by cleavage of the organometallic
bioconjugate from the resin, which generated active scaffolds
for hydrogenation reactions. This system shows characteristics
which mimics natural enzymes including a direct dependence of
the catalytic activity on proximal amino acid side chains, which
provides opportunities for evolution. While the heptapeptide
sequence is short to impose unique secondary structures,
molecular dynamics identified for some of the peptide
sequences clear energy minima. The established potential of
SPPS to prepare also longer structures and the availability of
mixed de-novo/ligation strategies offers approaches to
implement structural elements and to tailor further the
selectivity of this bio-inspired catalyst. A particularly appealing
aspect of the concept disclosed here is the opportunity to
combine an enzymatic scaffold with carbene complexes and
their vast array of catalytic applications. This combination
considerably expands the toolbox of biocatalysis and allows to
design systems for abiotic transformations.
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The authors gratefully acknowledge generous financial support
from the ERC (CoG 615653) and the Swiss National Science
Foundation (20020_182663 and 200021_192274).
Author Contributions
Author contributions: conceptualization (F.P., M.A.), data curation
(K.L., M.P., I.F., D.R.) data analysis, validation, visualization, and
writing (K.L., M.P., D.R. P., F.P., M.A).
There are no conflicts to declare.
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Notes and references
‡ Attempts to metallate the apo-peptides in the liquid-phase and
detached from the resin have failed so far, irrespective of whether
the heptapeptide was used or Fmoc-Hum without additional
amino acids.
§ Chiral GC analysis indicated that the benzyl alcohol was
obtained as a racemate in all catalytic runs, which can be
rationalized by the fact that the catalytic site is extremely exposed
in all modelled structures.
17 MD simulations were performed on the AXAHAXA sequence
with a natural His as the central amino acid (see ESI for
details). Replacing H with the Ḧ–Ir unit and rigidly fixed
atomic positions of the ligands and metal center for four
variants AXAḦAXA-Ir with X = K, H, W and Y did not reveal
any apparent changes in the behavior of the secondary
structure or solvent exposure (Figure S7).
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DOI: 10.1039/D1CC03158A
For Table of Contents use only
A non-natural histidinium amino acid has been developed and
used for solid-phase peptide synthesis to construct a peptide
iridium carbene conjugate as artificial mini-peptide for
hydrogenation catalysis.
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