A water soluble CuI-NHC for CuAAC ligation of unprotected peptides under open air conditions

Article abstract of DOI:10.1039/c2cc30515a

A reducing agent-free version of CuAAC able to operate under open air conditions is reported. A readily-synthesizable, hydrophilic and highly stable Cu^I-NHC allows the clean ligations of unprotected peptides comprising sensitive side chains, at

Full text of DOI:10.1039/c2cc30515a

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A water soluble CuI–NHC for CuAAC ligation of unprotected peptides
under open air conditionsw
Christelle Gaulier,a Audrey Hospital,a Bertrand Legeret,a Agnes F. Delmas,b
Vincent Aucagne,b Federico Cisnettia and Arnaud Gautier*a
Received 23rd January 2012, Accepted 28th February 2012
DOI: 10.1039/c2cc30515a
A reducing agent-free version of CuAAC able to operate under
open air conditions is reported. A readily-synthesizable, hydro-
philic and highly stable CuI–NHC allows the clean ligations of
unprotected peptides comprising sensitive side chains, at millimolar
concentrations.
The copper-catalyzed azide–alkyne cycloaddition (CuAAC)
reaction1 is considered as a standard technique for bioconjugation.2
This reaction requires that the catalytically active copper species,
often prepared from CuII, is brought or maintained in its air-
Fig. 2 Stable CuI–NHCs catalysts.
sensitive +I oxidation state. Most CuAAC bioconjugation
protocols3 combine an excess of an in situ reducing agent
(ascorbic acid,3a TCEPz or electrochemical reduction3b) with a
with some side chains if no precaution such as addition of
aminoguanidine is taken.3a
CuI nitrogen ligand3a–c (Fig. 1) and/or a drastic exclusion of
oxygen under glovebox-like conditions.3d In practice, the
triazole ligation of unprotected peptides or proteins can be
hampered by an exacerbated vulnerability to oxidation of some
side chains under standard CuAAC conditions. In this respect
methionine3d and histidine3a are particularly sensitive presumably
due to a sequence-dependent binding of copper to the sulfide or
imidazole, prior to the oxidation into sulfoxide and imidazolone,
respectively. Traces of O2 lead to reactive oxygen species, a
problem most pregnant under dilute conditions.3c This calls for
demanding experimental conditions that bring the experimenter
quite far away from some fundamental aspects of the ‘‘click’’
concept associated with this popular reaction. Moreover, ascorbic
acid degradation products can undergo deleterious reactions
These limitations led us to hypothesize that a reducing
agent-free catalytic system based on a water-soluble catalyst that
strongly stabilizes CuI should be envisaged. We were attracted by
the high stability of the copper(I) N-heterocyclic carbenes
(NHCs) [CuX(SIMes)] (1)z and [Cu(ICy)2]+ (2)z (Fig. 2).4a,b
Unfortunately, they are efficient for catalyzing azide–alkyne
cycloaddition only under neat or ‘‘on water’’ conditions. The
scope of the [CuCl(SIMes)] catalyst was increased by the
addition of aromatic N-donors, but 3a,b are still restricted to
alcoholic solvents.4c,d Recently Wang et al. reported the water
soluble catalyst 4, but it was still used ‘‘on water’’.4e In this
communication we report the scope and the limitations of the
new functionalized aqueous-soluble CuI–NHC, 5, allowing
clean CuAAC reactions in water with peptides bearing sensitive
side chains. To this end, a modification of the SIMes core by two
triazolyl-choline arms as hydrophilic and cationic surrogates
would fit the desired requirements of both stability and aqueous
solubility (Scheme 1).5 9 was obtained from 87 using the
stable catalyst 3b which allows a clean completion at room
temperature in an open flask. Therefore, 9.4HCl was refluxed
in ethanol–triethyl orthoformate, then in water, affording 10a.
A chloride-to-iodide exchange ensues to deliver the filterable
solid 10b. Finally, the copper atom was efficiently introduced
by the reaction with a stoichiometric amount of CuI in the
presence of NaOH in methanol to afford the heteroleptic
complex 5.y The choice of iodide anion was dictated by the
poor stability toward air oxidation of the corresponding
chloride complex whereas 5 was indefinitely stable upon
storage on the bench. The whole process is very practical
Fig. 1 Classical ligands used for CuAAC.
a Institut de Chimie de Clermont-Ferrand, CNRS UMR 6296,
Clermont-Universite´, 24 Ave des Landais, F-63177 Aubie`re, France.
E-mail: arnaud.gautier@univ-bpclermont.fr;
Fax: +33 (0)4 73 40 77 17; Tel: +33 (0)4 73 40 76 46
b
´
Centre de Biophysique Moleculaire, CNRS UPR 4301,
Rue Charles Sadron, F-45071 Orleans cedex 2, France
´
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc30515a
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4005–4007 4005

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Table 2 Influence of additives during the formation of 13
Entry
Additivea
Conversionb (%)
1
2
3
4
5
6
MeOH (25% v/v)
DMSO (25% v/v)
NMPz (25% v/v)
HFIPz (25% v/v)
NaCl (0.1 M)
100
100
100
100
80
MeCN (25% v/v)
50
a
In 0.2 M HEPES pH 7.6; [11] = 24 mM; [12] = 48 mM; [5] = 0.48 mM;
b
reaction time: 16 h; reactions were run in air. By H NMR.
1
Scheme 1 Synthesis of CuI–NHC 5. Reagents and conditions:
(a) Ref. 6, 93%. (b) NaN3, CuI (20 mol%), MeNH(CH2)2NHMe
(30 mol%), sodium ascorbate (20 mol%), DMSO : H2O (v/v) 9 : 1, 70 1C,
81%. (c) HO(CH2)2N(Me)2CH2CRCH, Cl, 3b (0.6 mol%), MeOH, then
HCl, 81%. (d) EtOH/HC(OEt)3, reflux, then water reflux, 10a: 74%,
then halogen exchange, 10b: 71%. (e) 5: CuI, NaOH, MeOH, 98%.
Table 3 Formation of 13 in the presence of L-amino acids
Entry L-Amino acida
5b Buffer (pH)c Conversiond (%)
1
2
3
4
5
6
7
8
Alanine
Cysteine
Glutathione
N-Acetyl alanine
N-Acetyl cysteine
N-Acetyl methionine
N-Acetyl histidine
N-Acetyl histidine
N-Acetyl histidine
N-Acetyl histidine
2
2
2
2
2
2
2
2
2
5
HEPES (7.6)
HEPES (7.6)
HEPES (7.6)
HEPES (7.6) 100
HEPES (7.6) 100
HEPES (7.6) 100
HEPES (7.6) B5
MES (6.2)
MES (5.7)
MES (6.2)
0
0
0
40
70
100
Scheme 2 CuAAC reaction between 11a and 12.
9
10
as it requires only simple filtrations and it is achievable on the
gram scale (ESIw).
a
b
c
20 mol%. mol%. [Buffer] = 0.2 M; [11] = 24 mM; [12] = 48 mM;
reaction time: 16 h; reactions were run under air. By 1H NMR.
d
We examined the ability of 5 to catalyze the formation of
13 from (S)-2-azido 3-(4-hydroxyphenyl) propanoic acid
(N3Tyr-OH, 11a)8 and propargyl alcohol (12) (Scheme 2) in
common aqueous buffers (pH: 10 to 5, Table 1) under
challenging conditions: low concentrations, air atmosphere,
low catalyst loading.
This screening allowed selecting HEPES and MES9
(entries 5 and 9) as the most suited buffers for our challenging
conditions. Also, pure water can be used (entry 1), which could
be advantageous for some synthetic purposes. We decided to
keep 0.2 M HEPES, pH 7.6 (entry 5) as standard conditions,
and investigated the effect of the addition of co-solvents or
salts (Table 2).
unprotected L-amino acids and glutathione, a tripeptide containing
an a-amino acid unit (Table 3). Clearly a-amino acids behave
as strong inhibitors (entries 1–3), whereas satisfactory results
were obtained with a tenfold excess (compared to the catalyst)
of N-acetylated derivatives: a full conversion took place in the
presence of N-acetyl alanine, cysteine and methionine (entries 4–6).
A lack of reactivity was observed at pH 7.6 with N-acetyl
histidine (entry 7). Nevertheless, this inhibition seems pH-dependent
as 40% and 70% conversions were reached by lowering the
pH to 6.2 and 5.7 (0.2 M MES buffer, entries 8 and 9),
respectively. The inhibition was finally suppressed using 5 mol%
of 5 (0.25 equiv./His) at pH 6.2 (entry 10).
Catalyst 5 was found to be compatible with methanol,
DMSO, NMP and HFIP (entries 1–4) whereas the presence
of chloride ions slightly decreased the conversion (entry 5).
Acetonitrile displays a stronger detrimental effect (entry 6). It
is noteworthy that chloride and MeCN have been reported as
inhibitors of THPTA/CuI-catalyzed CuAAC.3e As a-amino
acids usually chelate copper cations, we tested the influence of
Histidine side chain’s imidazole is known to participate in
the chelation of several metals through N1 binding. As the
imidazole’s H2 chemical shift is a good indicator of the
protonation state (protonated form: d = 8.40 ppm, basic form
d = 7.45 ppm),10 we examined the aromatic 1H NMR signature
of Na-acetylated histidine and found that H2 appears at 8.0 ppm
at pH 7.6 (0.2 M HEPES) and 8.4 ppm at pH 6.2 (0.2 M MES),
confirming, respectively, a partial and a total protonation
(ESIw). Thus, protonation appears to protect catalyst 5
from imidazole inhibition. The pH effect on the kinetics was
examined by 1H NMR (Fig. 3). Clearly, 13 forms more rapidly
in slightly basic (B3 h at pH 7.6) rather than acidic media
(>15 h at pH 6.2).
Table 1 Formation of 13 in buffered mediaa
Entry
Buffera
pH
Conversionb (%)
1
2
3
4
5
6
7
8
9
10
Nonec
Carbonate
TEABCz
Borate
N.D.d
10.2
8.8
7.9
7.6
7.6
7.4
7.4
6.2
95
0
45
10
100
0
0
0
100
0
HEPESz
TRISꢀmaleatez
TRISꢀHClz
Phosphate
MESz
Inspection of the initial curves also highlights the existence
of an induction period indicating that the catalytic species is
probably not 5. As the induction period decreases with
increasing pH, we hypothesize that the hydrolysis of the
copper-iodide bond is of importance in this process.
Acetate
4.7
a
[Buffer] = 0.2 M; [11] = 24 mM; [12] = 48 mM; [5] = 0.48 mM;
To prove the applicability of 5 to peptide synthesis, the CuAAC
ligation of model pentapeptides comprising residues sensitive
to copper-mediated oxidation (His, Met, Cys) was examined.
b
reaction time: 16 h; reactions were run in air. By H NMR. Using
d
the monosodium salt of 11a. Not determined.
1
c
c
4006 Chem. Commun., 2012, 48, 4005–4007
This journal is The Royal Society of Chemistry 2012

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other protective additives. Ligations are performed at low
concentrations under open air conditions on oxidation-sensitive
peptides, i.e.: containing methionine, cysteine, arginine, tyrosine,
tryptophan and histidine. The applicability of this methodology
to other bio(macro)molecules is currently studied. We believe
that our preliminary results pave the way for a new genera-
tion of CuAAC catalysts that will make this cycloaddition
a user-friendly tool for the bioconjugation of peptides and
proteins.
We thank Philippe Marceau for synthesizing the peptides.
Notes and references
Fig. 3 Kinetics of the formation of 13 at pH 6.2 and 7.6 (ESIw).
z SIMes = 1,3-bis(2,4,6-trimethylphenyl)imidazolin-2-ylidene; Icy =
1,3-bis(cyclohexyl)imidazol-2-ylidene; TEABC: triethylammonium
bicarbonate; HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic
acid; TRIS: tris(hydroxymethyl)methylamine; MES: 2-(N-morpholino)-
ethanesulfonic acid; NMP: N-methyl pyrrolidone; HFIP: hexafluoroiso-
propanol; TCEP: tris(2-carboxyethyl)phosphine.
y Our result contrasts with what was reported with the parent SIMes
and IMes ligands which afford a mixture of homo and heteroleptic
complexes (23/77).12 Steric hindrance and/or charge repulsion in 5
could account for this result.
1 (a) C. W. Tornøe and M. Meldal, in Peptides—The wave of the
future, ed. M. Lebl and R. A. Houghten, Kluwer Academic
Publishers, Dordrecht, 2001, pp. 263–264; (b) H. C. Kolb,
M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed., 2001,
40, 2004–2021; (c) C. W. Tornøe, C. Christensen and M. Meldal,
J. Org. Chem., 2002, 67, 3057–3064; (d) M. Meldal and C. W.
Tornøe, Chem. Rev., 2008, 108, 2952–3015.
Scheme 3 Catalyzed reactions of peptides in 0.2 M HEPES, pH 7.6.
2 R. K. V. Lim and Q. Lin, Chem. Commun., 2010, 46, 1589–1600.
3 (a) V. Hong, S. I. Presolski, C. Ma and M. G. Finn, Angew. Chem.,
Int. Ed., 2009, 48, 9879–9883; (b) V. Hong, A. K. Udit, R. A. Evans
and M. G. Finn, ChemBioChem, 2008, 9, 1481–1486; (c) A. Kumar,
K. Lib and C. Cai, Chem. Commun., 2011, 47, 3186–3188; (d) I. E.
Valverde, F. Lecaille, G. Lalmanach, V. Aucagne and A. F. Delmas,
Angew. Chem., Int. Ed., 2012, 51, 718–722; (e) S. I. Presolski,
V. Hong, S.-H. Cho and M. G. Finn, J. Am. Chem. Soc., 2010,
132, 14570–14576.
4 (a) S. Dıez-Gonzalez, A. Correa, L. Cavallo and S. P. Nolan,
Chem.–Eur. J., 2006, 12, 7558–7564; (b) S. Dıez-Gonzalez and S. P.
Nolan, Angew. Chem., Int. Ed., 2008, 47, 8881–8884; (c) M. L.
Teyssot, A. Chevry, M. Traıkia, M. El-Ghozzi, D. Avignant and
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and F. Li, Green Chem., 2011, 13, 3440–3445.
The three alkyne-containing peptides 14–16 were reacted at
a dilute 1 mM concentration with five fold excess of the
azides N3Tyr-OH (11a) or (S)-2-azido-3-phenyl-propanoic
acid (N3Phe–OH, 11b) in a 8–2 mixture of 0.2 M HEPES,
pH 7.6 and HFIP (Scheme 3).
The reactions were run overnight in an open flask at RT,
then worked up through a C18 solid phase extraction column
and analyzed by standard reverse phase HPLC/HRMS (ESIw).
The reaction of thiol-containing 14 gives a full conversion to the
triazole products using either 40 or 100 mol% of 5. As expected
from a long stay at neutral pH under non-deoxygenated
conditions, the products were obtained as the homodimeric
disulfides, but no over-oxidation into disulfide oxide, sulfenic
or sulfonic acids could be detected. Therefore, treatment of the
crude mixture with 5-fold excess of TCEP cleanly delivers the
expected products 14a,b (ESIw). The conversion of sulfide-
containing 15 into 15a,b was also clean and complete using
both 40 and 100 mol% of catalyst and no oxidation to the
methionine sulfoxide was observed. Regarding imidazole-
containing 16, an incomplete reaction occurred using 40 mol%
of catalyst in 0.2 M HEPES, pH 7.6 (B60% conversion).
Nevertheless, a total conversion was reached with one full
equivalent of 5 to afford 16a,b (ESIw). No trace of oxidation into
the imidazolone commonly observed under standard CuAAC
catalytic systems was detected.3a The reaction also applies quanti-
tatively to peptide 17 (ESIw), which corresponds to the N-terminal
octapeptide repeat region of the human prion protein (PrPc)
sequence and is known to strongly bind CuII.11 This proves that
good copper(II) chelators do not inhibit CuAAC mediated by 5.
In conclusion we report the first soluble copper(I)–NHC
that can realize CuAAC reactions with peptides in buffered
aqueous media in the absence of sacrificial reducing agents and
5 For
a review on hydrophilic catalysts: K. H. Shaughnessy,
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6 S. Leuthaeuber, V. Schmidts, C. M. Thiele and H. Plenio,
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7 J. Andersen, U. Madsen, F. Bjorkling and X. F. Liang, Synlett,
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9 HEPES and MES were originally designed to minimize metal
chelation, see: N. E. Good, G. D. Winget, W. Winter,
T. N. Connolly, S. Izawa and R. M. M. Singh, Biochemistry,
1966, 5, 467–477.
10 E. L. Ash, J. L. Sudmeier, R. M. Day, M. Vincent, E. V. Torchilin,
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12 S. Dıez-Gonzalez, E. C. Escudero-Adan, J. Benet-Buchholz,
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4005–4007 4007