Access to biomolecular assemblies through one-pot triple orthogonal chemoselective ligations

Article abstract of DOI:10.1002/anie.201006867

Three into one will go: The consecutive combination of three orthogonal chemoselective reactions (oxime ligation, thioether addition, and copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC)) in a sequential one-pot approach allows the syntheses of highly sophisticated biomolecular compounds without intervening isolations and protection schemes (see picture; ODN=oligodeoxynucleotide).

Full text of DOI:10.1002/anie.201006867

DOI: 10.1002/anie.201006867
Biomolecular Synthesis
Access to Biomolecular Assemblies through One-Pot Triple
Orthogonal Chemoselective Ligations**
Mathieu Galibert, Olivier Renaudet, Pascal Dumy, and Didier Boturyn*
The adequate combination of chemoselective ligations is
essential to have access to sophisticated biomolecular assem-
blies, thereby circumventing the problems associated with the
incompatible chemistries of carbohydrates, peptides, and
nucleic acids. In this regard, recent advances in the field of
chemoselective ligation have proven to be particularly
interesting for the construction of large biomolecular systems
for biological applications.[1] Since the use of a single chemo-
selective ligation limits the design complexity of a macro-
molecule, new methods for the ligation of multiple biomo-
lecular partners are required. The development of chemo-
selective ligations of unprotected peptides enables the routine
chemical synthesis of proteins.[2] Very recently, Carell et al.
have used the copper(I)-catalyzed alkyne–azide cycloaddition
(CuAAC)[3] to label nucleic acids with three different tags.[4]
A critical point is the requirement of orthogonal protecting
groups and purification steps. Ideally, sophisticated biomo-
lecular assemblies can be generated by a combination of
orthogonal chemoselective ligations. Recently, we described
the combination of CuAAC and orthogonal oxime bond
formation, which allows the one-pot synthesis of peptide
conjugates.[5] Other groups have reported the combination of
two orthogonal chemoselective ligations for the synthesis of
neoglycopeptides,[6] and for the labeling of peptides.[7] Access
to more sophisticated biomolecular compounds remains a
challenging task.
Herein, we report that this chemistry can be extended to
prepare sophisticated compounds encompassing a diversity of
biomolecules, such as peptides, sugars, and nucleic acids
(Scheme 1). The way to achieve such molecular assemblies is
to use one-pot triple orthogonal chemoselective ligations
through the combination of the CuAAC reaction, oxime
ligation, and thioether bond formation. This methodology
enables the ligation of up to four different molecular frag-
ments without the requirement of protection schemes and/or
tricky intermediate purifications. To illustrate this strategy, we
constructed multifunctional Arg-Gly-Asp (RGD)-containing
Scheme 1. Structure of the RGD-containing biomolecular system. y:
triazole, oxime; c: oxime, thioether; z: thioether, triazole; R: peptide,
nucleic acid, or dye.
biomolecular systems encompassing a carbohydrate residue
along with a molecular fragment that can be used for
diagnostics (fluorescent probe) or for therapy (peptide or
nucleic acids; see Scheme 2). The benefit of the RGD-
containing cyclodecapeptides for tumor targeting was pre-
viously demonstrated,[8] and recent research efforts have
shown that additional incorporation of a carbohydrate moiety
within the RGD-containing compounds provided an
enhanced compound solubility and clearance.[9]
To test the feasibility of our approach we first prepared a
cyclodecapeptide scaffold 1 containing aldehyde, alkyne, and
maleimide groups, which allowed the fully convergent syn-
thesis of the RGD-containing neoglycopeptides 2 (Sche-
me 2a). To introduce aldehyde and alkyne functions, we
ꢀ
incorporated the building blocks N-Fmoc-Lys[CO-(CH2)2-C
CH] (Fmoc = 9-fluorenylmethoxycarbonyl) and N-Fmoc-
Lys[N-Boc-Ser(O-tBu)] (Boc = tert-butoxycarbonyl) into the
decapeptide chain by using standard solid-phase peptide
synthesis (SPPS; see the Supporting Information). These
building blocks are very important because they considerably
reduce the number of steps involved and the combination of
protecting groups required for the synthesis of the function-
alized peptides. As the maleimide function reacts rapidly with
piperidine, which is commonly used during SPPS, the thiol-
reactive maleimide group was consequently added at a lysine
side chain after SPPS (see the Supporting Information). In
parallel, the cyclopentapeptide cyclo[-Arg-Gly-Asp-DPhe-
Lys(-CO-CH2-N3)-] 3 bearing the prerequisite azide function
was prepared through a combination of solid- and solution-
[*] M. Galibert, Dr. O. Renaudet, Prof. Dr. P. Dumy, Dr. D. Boturyn
Dꢀpartment de Chimie Molꢀculaire
UMR CNRS/UJF 5250, ICMG FR 2607
570 rue de la chimie, BP53, 38041 Grenoble cedex 9 (France)
Fax: (+33)4-5652-0805
E-mail: didier.boturyn@ujf-grenoble.fr
[**] This work was supported by the Universitꢀ Joseph Fourier, the
Centre National de la Recherche Scientifique (CNRS), the Institut
National du Cancer (INCA), and the Nanoscience Foundation. We
are grateful to NanoBio (Grenoble) for the facilities of the Synthesis
Platform.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201006867.
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Communications
Scheme 2. a) Reagents and conditions for the synthesis of compounds 2: 1) R1-ONH2 4 (3 equiv), CH3CN/PBS (pH 2.2, 40:60), 1 h; 2) R2-SH 6
(1.3 equiv), pH adjusted to 7 using NaHCO3, 30 min; 3) R3-CH2-N3 3 (6 equiv), Cu0 (5 equiv), tBuOH. b) Reagents and conditions for the
synthesis of compounds 9: 1) lactose (5 equiv), CH3CN/ammonium acetate buffer (pH 4.5, 40:60), 508C, 5 h; 2) Alexa Fluor 647 C2-maleimide
(1.3 equiv), pH adjusted to 7 using NaHCO3, 30 min; 3) R3-CH2-N3 3 (6 equiv), Cu0 (5 equiv), tBuOH. c) Reagents and conditions for the
synthesis of compounds 12: 1) R3-CHO 15 (5 equiv), TFA/CH3CN/H2O (5:45:50), 1 h; 2) pH adjusted to 7 using NaHCO3, R1-SH 16 (2 equiv),
6
0
ꢀ
30 min; 3) R -C CH 13 (0.8 equiv), Cu (5 equiv), tBuOH, 508C, 5 h. PBS=phosphate-buffered saline, TFA=trifluoroacetic acid.
phase syntheses.[5] Glc-b-ONH2 4 was prepared by glycosyla-
tion of N-hydoxysuccinimide using glycosyl fluoride as donor
and BF3·Et2O as promoter.[10]
successfully within 1 hour the glycopeptide intermediate 5
(Figure 1a). The pH was then adjusted to neutral conditions
and KLA peptide 6[11] (1.3 equiv) bearing an N-terminal
cysteine residue was added to the reaction mixture. Rapid
thiol addition was observed at room temperature providing
within 30 minutes the intermediate 7 (Figure 1a). It is worth
mentioning that under neutral or acidic conditions, the
addition of the remaining aminooxy-carbohydrate 4 (ca.
2 equiv) to the maleimide moiety was not detected. The
subsequent third chemoselective ligation was carried out by
adding Cu0 microsize powder (5 equiv)[12] and azido RGD-
peptide 3 (1.5 equiv per site) to the reaction mixture. The
alkyne–azide cycloaddition furnished the expected RGD-
With the trifunctional compound 1 in hand, we then
carried out the one-pot sequential chemoselective ligations of
the different biomolecules (Scheme 2a). We decided to
perform the CuAAC after oxime ligation and thiol addition
as the aldehyde residue is not stable under the CuAAC
conditions,[9] and oxidation of thiopeptides was observed in
the presence of copper. Chemoselective ligations were care-
fully monitored by HPLC analysis (Figure 1). Addition of
Glc-b-ONH2 4 (3 equiv) to the aldehyde-containing cyclo-
peptide 1 was carried out under acidic conditions providing
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Figure 1. a) Chemoselective assembly of compound 2. HPLC profiles (214 nm) for 1) scaffold 1, and for 2) oxime ligation of carbohydrate 4,
3) thioether addition of peptide 6, and 4) CuAAC of peptide 3. b) Chemoselective assembly of compound 9. HPLC profiles (214 nm) for 1) scaffold
8, and for 2) oxime ligation of lactose, 3) thioether addition of Alexa Fluor 647, and 4) CuAAC of peptide 3. c) Chemoselective assembly of
compound 13. HPLC profiles (260 nm) for 1) free oligonucleotide 13 and 2) CuAAC of oligonucleotide 13 to intermediate 18.
containing neoglycopeptide 2 in yields of about 55% after
purification. During CuAAC, KLA dimer that results from
the oxidation of the residual thiopeptide 6 was detected.
To allow the grafting of a reducing sugar, we decided to
exploit an alternative one-pot approach allowing the synthesis
of the fluorescent neoglycopeptide 9 (Scheme 2b). We then
prepared the scaffold 8 containing aminooxy, alkyne, and
thiol groups. These functions are easily incorporated within
the peptide during the SPPS (see the Supporting Informa-
azido function was prepared by means of N-Fmoc-e-azido-
norleucine. The latter was obtained from the diazo transfer of
imidazole-1-sulfonyl azide to N-Fmoc-lysine.[17] Aminooxy
functions and the iodoacetamide group were chosen to graft
respectively the homing peptide cyclo[-Arg-Gly-Asp-DPhe-
Lys(-CO-CHO)-] 15 and the b-d-thioglucose 16. With the
scaffold 14 in hand, we then carried out the one-pot sequential
chemoselective ligations of peptide 15, sugar 16, and oligo-
nucleotide 13 (Scheme 2c). As the nucleic acids are sensitive
to acidic conditions, we first performed simultaneous Eei
removal and oxime ligation of the aldehyde-containing
peptide 15.[13] The resulting RGD peptide intermediate 17
was then converted to the neoglycopeptide 18 under neutral
conditions (see the Supporting Information). Finally, the
ligation of the oligonucleotide 13 and the intermediate 18 was
obtained through CuAAC (Figure 1c). Reversed-phase
HPLC methods furnished the expected biomolecular com-
pound 12 in yields of about 60%. This result emphasizes the
utility of this method for the synthesis of complex biomolec-
ular assemblies.
In conclusion, we have shown for the first time that
consecutive combination of three chemoselective reactions
could be carried out in a one-pot approach. This strategy
allows efficient and rapid syntheses of biomolecular com-
pounds without intervening isolations and protection
schemes. We believe that this strategy should pave the way
for multifunctionalized macromolecular systems with tailor-
made properties. To design immunoactive glycoconjugates,[18]
we are currently implementing this strategy for the construc-
tion of a new generation of synthetic vaccine prototypes
combining multiple cancer antigens within the same mole-
cule. Immunological evaluation will be reported in due
course.
=
tion). N-Fmoc-Lys[-CO-CH2-O-N C-CH3(OEt)] including 1-
ethoxyethylidene (Eei) as protecting group for the aminooxy
moiety was preferred because the Eei group is fully compat-
ible with standard SPPS conditions.[13] Starting with the
functionalized scaffold 8, we first performed the ligation of
lactose (Scheme 2b). Oxime formation was achieved with
exclusive specificity under mild acid conditions after 5 hours
at 508C (Figure 1b). Taking note of a recent report,[14] further
investigation revealed that the aniline Schiff base (100 mm)
decreased the reaction time to 1 hour. However, this reagent
was found to be incompatible with the next ligations. Rapid
aniline addition to maleimide was observed. The resulting
glycopeptide 10 was subsequently converted to the fluores-
cent intermediate 11 simply by adjusting the pH to neutral
conditions and by adding the maleimide-containing Alexa
fluorescent probe. After 30 minutes, the orthogonal thiol
addition to maleimide was complete (Figure 1b). The last
ligation of the azido RGD-peptide 3 and the alkyne-contain-
ing intermediate 11 furnished the desired triply modified
compound 9. The fluorescent compound 9 was readily
purified by reversed-phase HPLC methods and characterized
by mass spectrometry (see the Supporting Information).
To demonstrate the broad utility of our method, we
decided to extend the scope of this approach to nucleic acids.
A last strategy was then adopted for the synthesis of the
biomolecular conjugate 12 (Scheme 2c). Numerous works
have demonstrated the effectiveness of the CuAAC reaction
for oligonucleotide conjugation.[15] For this reason, we syn-
thesized the alkyne-containing oligonucleotide 13[16] by using
the commercially available hexyn-5-yl phosphoramidite. In
parallel, the scaffold 14 encompassing the complementary
Received: November 2, 2010
Published online: January 12, 2011
Keywords: biomolecules · chemoselectivity · click chemistry ·
.
cycloaddition · ligation
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