Protected maleimide building blocks for the decoration of peptides, peptoids, and peptide nucleic acids

Article abstract of DOI:10.1021/bc4000614

Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides into polyamides such as peptides, peptide nucleic acids, and peptoids were prepared, as well as the corresponding oligomers. Suitable maleimide deprotection conditions were

Full text of DOI:10.1021/bc4000614

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Technical Note
Protected maleimide building blocks for the decoration
of peptides, peptoids and peptide nucleic acids
Anna Grandas, Xavier Elduque, Albert Sanchez, Kapil Sharma, and Enrique Pedroso
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/bc4000614 • Publication Date (Web): 14 Apr 2013
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PROTECTED MALEIMIDE BUILDING BLOCKS FOR THE DECORATION
OF PEPTIDES, PEPTOIDS AND PEPTIDE NUCLEIC ACIDS
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Xavier Elduque,# Albert Sánchez,# Kapil Sharma, Enrique Pedroso and Anna Grandas*
Albert Sánchez, Enrique Pedroso and Anna Grandas*
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Departament de Química Orgànica and IBUB, Facultat de Química, Universitat de
Barcelona; Martí i Franquès 1-11, 08028 Barcelona, Spain
Phone: + 34934021263
Fax: + 34933397878
e-mail: anna.grandas@ub.edu
#These two authors contributed equally to this work.
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ABSTRACT
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Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides
into polyamides such as peptides, peptide nucleic acids and peptoids were prepared, as
well as the corresponding oligomers. Suitable maleimide deprotection conditions were
established in each case. The stability of the adducts generated by Michael-type
maleimide-thiol reaction and Diels-Alder cycloaddition to maleimide deprotection
conditions was exploited to prepare a variety of conjugates from peptide and PNA
scaffolds incorporating one free and one protected maleimide. The target molecules
were synthesized by using two subsequent maleimide-involving click reactions
separated by a maleimide deprotection step. Carrying out maleimide deprotection and
conjugation simultaneously gave better results than performing the two reactions
subsequently.
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INTRODUCTION
Controlled ligation of different building blocks provides access to complex structures,
libraries of products, and biomolecules modified à la carte. Biocompatible materials,1,2
polymers with pharmacological application,3 new drugs with higher affinity and
specificity for the target,4,5 biomolecules with optimized properties (increased half life
in biological fluids, enhanced uptake, incorporating reporter groups for imaging, etc.),6
and complex nanostructures and supramolecular systems7,8 have been prepared taking
advantage of those chemical tools.
In the past few years different alternatives have been described for the modular
construction of sophisticated molecules making use of so-called "click" reactions.9 One
of the options consisted in attaching different alkynes to a scaffold, either combinations
of strained and terminal alkynes,10 or of unprotected and orthogonally protected
alkynes.11-14 Subsequent Huisgen reactions with azides, with or without Cu(I) or after
the required deprotection treatment, allowed new building blocks to be placed where
desired.
Other experiments also involving Cu(I)-catalyzed Huisgen cycloadditions15-17 have
made use of a scaffold with two different appending groups, an azide and either a group
that can be transformed into azide or a latent 1,3-dipole. The alkyne-azide reaction was
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followed by functional group conversion (that is, transformation of the azide precursors
into azides or of the latent 1,3-dipole into dipole), and this by the second cycloaddition.
Chemoselective reactions have been subsequently performed on scaffolds derivatized
with two or three different functional groups. Huisgen cycloadditions, oxime and
hydrazone formation, and thiol-maleimide reactions have been some of the most
commonly chosen orthogonal reactions.18-20 The thiol-ene and inverse electron-demand
Diels-Alder reactions are becoming increasingly popular and have also been used in
combination with other chemoselective processes.21-23
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Finally, a combination of stepwise synthesis and attachment of different building blocks
at different elongation stages also has allowed different moieties to be attached to a
polyamide chain.24,25
Maleimides are versatile molecules susceptible to be used in "click" reactions with
thiols, as already mentioned, and with dienes. Unprotected maleimides are not
compatible with basic, nucleophilic reagents, which may promote maleimide hydrolysis
and/or add to the double bond. We have recently described that the exo cycloadduct
obtained by reaction of an N-alkylated maleimide with 2,5-dimethylfuran is stable to
concentrated aqueous ammonia at room temperature, which can be exploited for the on-
resin synthesis of maleimido-oligonucleotides.26,27 5'Maleimido-oligonucleotides were
prepared by coupling derivatives incorporating protected maleimide cycloadducts
followed by deprotection with ammonia and retro–Diels-Alder reaction.
The goal of this work was the preparation of maleimido-containing peptides, peptide
nucleic acids (PNAs) and peptoids, and their use in conjugation reactions (Scheme 1).
Scheme 1.
Solid-phase synthesis of these polyamides requires reaction with bases, either to remove
the Fmoc (9-fluorenylmethoxycarbonyl) temporary protecting group in the case of
peptides and PNAs, or to incorporate the construction units during peptoid assembly
using the submonomer strategy.28 Therefore, use of [2,5-dimethylfuran]-protected
maleimide building blocks was expected to allow introduction of a maleimide unit into
any position of the chain.
The first step was the preparation of monomers incorporating the protected maleimide
moiety (Scheme 2), which were subsequently introduced into the corresponding
oligomers. Maleimide deprotection provided fully reactive maleimides in all cases, as
assessed by reaction with thiol- or diene-containing compounds (Scheme 3).
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Assessment of the stability of the adducts generated after Michael-type and Diels-Alder
reactions to maleimide deprotection conditions prompted us to attach protected and
unprotected maleimides to the same polyamide scaffold (peptide, PNA), and use them
for two subsequent maleimide-involving click reactions (Scheme 4). This methodology
offers so far unexplored possibilities for the chemical preparation of libraries of
compounds.
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MATERIALS AND METHODS
General materials and methods (origin of products, procedures for solid-phase
polyamide assembly, and conditions for the analysis and characterization of the
different synthesized compounds) are described at the Supporting Information.
Scheme 2
[Protected maleimido]propanoic acid N-hydroxysuccinimido ester 2. To a solution
of 1 (1.04 g, 3.91 mmol) in DCM (dichloromethane, 7.5 mL), EDC·HCl (N-ethyl-N'-(3-
dimethylaminopropyl)-carbodiimide, 820 mg, 4.3 mmol) and N-hydroxysuccinimide
(490 mg, 4.3 mmol) was added. The mixture was stirred at room temperature for 84 h.
The reaction mixture was diluted with DCM to 75 mL, and the resulting solution was
washed with 0.1 M HCl (2 ×). The organic fraction was dried over anh. MgSO4 and
filtered, and the solvent was removed in vacuo. A white foam was obtained (1.23 g,
84%). The product was pure enough to be used in the next step. If required, 2 can be
purified by silica gel column chromatography eluting with a 1:1 mixture of
hexanes/AcOEt and increasing amounts of AcOEt (up to 100%).
1
Rf = 0.7 (AcOEt); H-NMR (400 MHz, CDCl3): δ 6.30 (s, 2H), 3.88 (t, J = 7.23 Hz,
2H), 2.98 (t, J = 7.24 Hz, 2H), 2.88 (s, 2H), 2.81 (s, 4H), 1.70 (s, 6H) ppm; 13C-NMR
(101 MHz, CDCl3): δ 174.4, 168.9, 165.8, 140.8, 87.6, 77.3, 52.5, 33.7, 28.8, 25.5,
15.7; HRMS (ESI, positive mode): m/z 363.1209 [M+H]+, 380.1441 [M+NH4]+,
747.2128 [2M+Na]+, M calcd. for C17H18N2O7 362.1114.
Nα-Fmoc-Lys(Nε-[protected maleimido]propanoyl)-OH 3. Nα-Fmoc-Lys-OH (254
mg, 0.69 mmol) was dissolved in a mixture of trifluoroethanol (8 mL), THF
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(tetrahydrofuran, 4 mL) and DIEA (N,N-diisopropylethylamine, 0.36 mL, 2.1 mmol). 2
(300 mg, 0.83 mmol) was added to the solution, and the mixture was stirred for 24 h at
room temperature, and for 5 days at 5 ºC. After the solvent was removed in vacuo, the
crude was dissolved in AcOEt and washed with a 10% aqueous solution of citric acid (3
×). The organic fraction was collected and dried over MgSO4 and filtered, and the
solvent was removed under reduced pressure. Lysine derivative 3 was purified by silica
gel column chromatography, eluting with AcOEt and 0.1% AcOH, and increasing
amounts of MeOH (up to 10 %). The appropriate fractions were pooled and evaporated
to dryness, to yield a white foam (292.6 mg, 69 %).
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Rf = 0.4 (AcOEt/MeOH/AcOH 10:1:0.1); IR (ATR), ν: 3345, 2934, 1691, 1529, 1444,
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1215, 1167 cm-1; H-NMR (400 MHz, CDCl3): δ 7.74 (d, J=7.54 Hz, 2H), 7.59 (t, J =
6.80 Hz, 2H), 7.38 (t, J = 7.45 Hz, 2H), 7.29 (t, J = 7.43 Hz, 2H), 6.26 (s, 2H), 4.37 (d,
J = 7.07 Hz, 2H), 4.20 (t, J = 6.90 Hz, 1H), 3.75 (t, J = 7.06 Hz, 2H), 3.22 (d, J = 5.73
Hz, 2H), 2.81 (s, 2H), 2.48 (t, J = 6.97 Hz, 2H), 1.66 (s, 6H), 2-0.8 (bs, 6H) ppm; 13C-
NMR (101 MHz, CDCl3): δ 143.7, 141.3, 140.8, 127.7, 127.1, 125.1, 120.0, 87.7, 67.0,
52.5, 47.1, 39.0, 35.1, 34.3, 29.7, 28.6, 15.8 ppm; MALDI-TOF MS (DHB=2,5-
dihydroxybenzoic acid, positive mode): m/z 638.3 [M+Na]+, 542.3 [M+Na-Me2furan]+,
520.3 [M+H-furan]+, M calcd. for C34H37N3O8 615.3. HRMS (ESI, negative mode): m/z
614.2481 [M-H]-, M calcd. for C34H37N3O8 615.2581.
N-[Protected maleimido]-N-(2-Fmoc-aminoethyl)glycine tert-butyl ester 4. N-(2-
Fmoc-aminoethyl)glycine tert-butyl ester·HCl (890 mg, 2.06 mmol) was dissolved in
DCM (120 mL) and washed with a saturated NaHCO3 aqueous solution (3 × 50 mL).
The organic solution was dried over anh. Na2SO4 and filtered, and the solvent was
removed in vacuo. The resulting yellowish oil (810 mg of N-(2-Fmoc-
aminoethyl)glycine tert-butyl ester, quantitative recovery) was dissolved in DMF (N,N-
dimethylformamide, 3 mL) and bubbled with Ar. To this solution was added a mixture
containing 1 (610 mg, 2.30 mmol), HOBt·H2O (1-hydroxybenzotriazole, 544 mg, 3.55
mmol),
HATU
(2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate, 873 mg, 2.30 mmol) and N-methylmorpholine (410 ꢀL, 3.73
mmol) previously stirred at room temperature under an Ar atmosphere for 20 min. The
resulting mixture was stirred for 60 h at room temperature under an Ar atmosphere.
Solvent was removed under reduced pressure, and the resulting crude was purified by
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silica gel column chromatography eluting with a 1:1 (v/v) hexanes/AcOEt mixture. 4
Was obtained as a pale yellowish oil (1.06 g, 80 % overall yield).
1H-NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.6 Hz, 2H), 7.61 (t, J = 8.4 Hz, 2H), 7.39
(t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 6.28 (s, 2H), 5.97 (t, J = 6.2 Hz) and 5.64 (t,
J = 5.4 Hz) (rotamers, 1H), 4.37 (m, 2H), 4.23 (m, 1H), 3.89 (s, 1H), 3.80 (m, 2H), 3.47
(m, 2H), 3.35 (m, 2H), 2.80 (s, 6H), 2.64 (t, J = 7.6 Hz), 2.52 (t, J = 7.1 Hz) (rotamers,
2H), 1.68 (s, 9H) ppm; 13C-NMR (101 MHz, CDCl3): δ 176.2, 166.1, 63.4, 61.7, 57.0,
43.9, 41.0, 36.7, 29.8, 29.5, 26.8, 24.5 ppm; HRMS (ESI, positive mode): m/z 644.2955
[M+H]+, 661.3227 [M+NH4]+, 666.2782 [M+Na]+, 1304.6093 [2M+NH4]+, 1309.5680
[2M+Na]+, M calcd. for C36H41N3O8 643.2887.
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N-[Protected maleimido]-N-(2-Fmoc-aminoethyl)glycine 5. A solution of 4 (500 mg,
0.777 mmol) in DCM (10 mL) was chilled in an ice bath. TFA (trifluoroacetic acid, 5
mL) and TIS (triisopropylsilane, 2 mL) was added, and the mixture was left to react at 0
oC for 30 min and then at room temperature until TLC analysis (hexanes/AcOEt 1:4, Rf
= 0.21) showed complete disappearance of 4 (3 h reaction time). Solvent and reagents
were removed in vacuo, and the resulting oil was dissolved in the minimal amount of
DCM and precipitated over cold hexanes. A white solid was obtained after decantation
(455 mg, quantitative yield), which was thoroughly dried in a desiccator before its use
in PNA synthesis.
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IR (ATR), ν: 3349, 2937, 1768, 1694, 1523, 1446, 1403, 1170 cm-1; H-NMR (400
MHz, CDCl3): δ 7.74 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 5.5 Hz, 2H), 7.38 (t, J = 7.3 Hz,
2H), 7.30 (t, J = 6.7 Hz, 2H), 6.27 and 6.26 (2s, 2H), 5.78 (brs) and 5.69 (brs)
(rotamers, 1H), 4.60 (brs) and 4.54 (brs) (rotamers, 1H), 4.37 (m, 2H), 4.21 (t, J = 6.4
Hz, 1H), 4.03 (m, 2H), 3.76 (brs, 4H), 3.49 (brs, 2H), 3.35 (brs, 2H), 2.80 (m, 2H), 2.68
(brs) and 2.55 (brs) (rotamers, 2H), 1.66 (s, 6H) ppm; 13C-NMR (101 MHz, CDCl3): δ
174.7, 172.6, 171.7, 156.7, 143.9, 141.3, 140.9, 127.7, 127.1, 125.1, 120.0, 87.6, 67.0,
52.5, 49.5, 49.2, 47.1, 39.4, 34.9, 30.1, 15.9 ppm; HRMS (ESI, positive mode): m/z
588.2328 [M+H]+, 605.2599 [M+NH4]+, 610.2159 [M+Na]+, 1175.4609 [2M+H]+,
1192.4849 [2M+NH4]+, 1197.4380 [2M+Na]+, M calcd. for C32H33N3O8 587.2261.
2,5-Dimethylfuran-protected maleimide (exo + endo) 6. Maleimide (3.0 g, 30.9
o
mmol) was dissolved in ACN (acetonitrile, 55 mL). The solution was heated to 60 C,
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and 2,5-dimethylfuran (8.3 mL, 77.3 mmol) was added. After overnight reaction at 60
oC, the mixture was evaporated to dryness to yield 6 as a 4:1 mixture of exo and endo
isomers.
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Exo isomer: H-NMR (400 MHz, CDCl3): δ 6.31 (s, 2H), 2.88 (s, 2H), 1.73 (s, 6H)
ppm; 13C-NMR (101 MHz, CDCl3): δ 175.0, 140.9, 87.7, 53.8, 15.8 ppm.
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Endo isomer: H-NMR (400 MHz, CDCl3): δ 6.31 (s, 2H), 3.27 (s, 2H), 1.78 (s, 6H)
ppm; 13C-NMR (101 MHz, CDCl3): δ 174.4, 138.2, 135.1, 54.9, 18.5 ppm.
HRMS (ESI, positive mode): m/z 194.0915 [M+H]+, M calcd. for C10H11NO3 193.0739.
N-Boc-ethanolamine 7.29 Ethanolamine (6.1 g, 0.1 mol) was added to Boc2O
o
(Boc=tert-butoxycarbonyl, 21.8 g, 0.1 mol) in three portions at 0 C over 10 min, and
the mixture was stirred for 2 h. A colorless precipitate formed with the evolution of
heat. After filtration, the solid was purified by silica gel column chromatography eluting
with a 6:1 hexanes/AcOEt mixture. The desired fractions (as assessed by TLC:
hexanes/AcOEt 2:3, Rf = 0.5, detection: reaction with anisaldehyde) were pooled and
taken to dryness, which yielded pure 7 as a colorless oil (15.8 g, 98 %).
1H-NMR (400 MHz, CDCl3): δ 5.05 (bs, 1H), 3.68 (m, 2H), 3.26 (t, 2H, J = 4.8 Hz),
2.83 (bs, 1H), 1.43 (s, 9H) ppm; 13C-NMR (101 MHz, CDCl3): δ 156.81, 79.63, 62.47,
43.10, 28.34 ppm.
N-[Protected maleimido]-ethanediamine·TFA 9 (exo). A mixture of protected
maleimide 6 (500 mg, 2.59 mmol), N-Boc-ethanolamine 7 (278 mg, 1.73 mmol) and
triphenylphosphine (679 mg, 2.59 mmol) was coevaporated with anh. THF, dissolved in
anh. THF (20 mL) and chilled in an ice bath. DIAD (diisopropylazodicarboxylate, 511
o
ꢀL, 2.59 mmol) was added dropwise. The mixture was reacted for 2 h at 0 C and
overnight at room temperature, and then the solvent was removed under reduced
pressure. The resulting residue (which contained the mixture of exo and endo isomers)
was treated with a 2:3 v/v MeOH/conc. aq. ammonia mixture overnight, and then
concentrated under reduced pressure. CHCl3 (20 mL) was added, and the pH of the
aqueous phase was adjusted to 12-13. The two phases were separated, and the organic
phase was reextracted with 0.01 M NaOH (20 mL, 3 ×), dried over MgSO4 and taken to
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dryness. H NMR analysis showed that the resulting residue contained 8 as the exo
isomer, plus byproducts resulting from the Mitsunobu reaction (triphenylphosphine
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oxide and DIADH2). Crude 8 was treated with a 3:7 TFA/DCM mixture (30 mL) for 1 h
at room temperature, and then was taken to dryness. The desired product precipitated
upon addition of anhydrous diethyl ether (2 mL). After centrifugation the supernatant
was discarded and diethyl ether was added again to the precipitate. This step was
repeated three times. The product was dried thoroughly under reduced pressure, to
afford pure 9 (119 mg, 20 %).
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8: H-NMR (400 MHz, CDCl3): δ 6.12 (s, 2H), 3.44 (t, J=5.61 Hz, 2H), 3.10 (m, 2H),
2.65 (s, 2H), 1.51 (s, 6H), 1.22 (s, 9H) ppm.
9: IR (ATR), ν: 3574, 2985, 1769, 1694, 1674, 1402, 1200, 1167, 1124 cm-1; 1H-NMR
(400 MHz, acetone-d6): δ 6.38 (s, 2H), 3.96 (t, J=5.33 Hz, 2H), 3.83 (t, J=5.36 Hz, 2H),
13
2.95 (s, 2H), 1.59 (s, 6H) ppm; C-NMR (101 MHz, acetone-d6): δ 176.8, 142.7, 89.2,
54.8, 46.3, 38.3, 17.2 ppm; HRMS (ESI, positive mode): m/z 473.2388 [M+H]+, M
calcd. for C24H32N4O6 472.2317.
Syntheses of the conjugates involved the following series of steps:
a: Mono-derivatized polyamides. i) Elongation of polyamide chains by solid-phase
synthesis, using commercially available building blocks and the protected maleimido
monomers here described (3, 5 and 9 were used to introduce the protected maleimide
moieties at any position within the chain, 3-maleimidopropanoic acid 1 was only used
for N-terminal derivatization); ii) acid treatment to remove all protecting groups except
that on the maleimide moiety; iii) maleimide deprotection following one of the
procedures described below; iv) conjugation (reaction with either a thiol or a diene, see
below).
b: Doubly-derivatized polyamides. Steps i) and ii) were the same as above; iii) first
conjugation (reaction of the free maleimide with either a thiol or a diene); iv) maleimide
deprotection; v) second conjugation (second reaction with either a thiol or a diene).
c: Alternatively, in some cases maleimide deprotection and conjugation were carried
out simultaneously (see below).
Microwave-promoted maleimide deprotection. This procedure was used to deprotect
maleimides appending from PNAs.
A solution (1000 ꢀL) of [protected maleimido]-containing PNA in a 1:1 (v/v)
MeOH/H2O mixture (25 ꢀM concentration) was introduced in a microwave vial and
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o
irradiated for 60 min at 100 C. Solvent was removed under vacuum, and the resulting
crude was dissolved in water for HPLC analysis and characterization by MALDI-TOF
MS. The crude was used at the subsequent conjugation step without any purification.
8
9
Maleimide deprotection by heating in toluene. This procedure was used to deprotect
maleimides appending from peptides and peptoids.
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The [protected maleimido]-containing peptide/peptoid was dried by coevaporation with
anh. toluene (3-4 ×), and a new batch of anh. toluene was added (the amount that would
be required to obtain a 25 ꢀM solution). The mixture was heated at 90 ºC, toluene was
removed under reduced pressure, and the crude was dissolved in water for HPLC
analysis and mass spectrometric characterization. The crude was used at the subsequent
conjugation step without any purification.
Conjugation: General procedure for Michael-type reactions. Aliquots of aqueous
(or aqueous-organic) solutions containing the required amounts of maleimido-
containing oligomer and the corresponding thiol-containing compound (5 to 10-fold
molar excess) were mixed, and the mixture was diluted with 0.5 M triethylammonium
acetate, pH = 7.8-7.9 (final concentration of oligomer: 50-150 ꢀM). The mixture was
stirred at room temperature under an Ar atmosphere. Initially all experiments were
carried out overnight; yet, we have verified that in most cases the reaction is complete
in 1 h or even less. Shortening the reaction time prevents hydrolysis of the succinimide
formed after the thiol-maleimide reaction, which has occasionally been observed
(compound 21). Reaction crudes were analyzed by HPLC. Conjugates were purified by
HPLC and characterized by MALDI-TOF MS.
When reagents were not soluble in water the reaction was carried out in organic solvent-
aqueous buffer mixtures (see Supporting information, synthesis of conjugate 22).
Conjugation: General procedure for Diels-Alder cycloadditions. The maleimido-
containing oligomer and the diene-oligonucleotide were dissolved in water (final
concentration of maleimido-containing oligomer: 50-250 ꢀM). The molar ratio varied
between 1:1 and 1:5, the component in excess generally being the least valuable one, in
other words, the most easily accessible (which of the two reagents is used in excess was
found to have no influence on the reaction yield - data not shown). The mixture was
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reacted at 37 C, generally overnight. Reaction crudes were analyzed by HPLC.
Conjugates were purified by HPLC and characterized by MALDI-TOF MS.
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One-pot [maleimide deprotection + conjugation] reactions. After coevaporation with
anh. toluene (3 ×), the protected maleimido-containing polyamide, and either the diene
(1-2 equiv) or the thiol-containing compound (10-fold molar excess) were dissolved or
suspended in anh. toluene (concentration of the polyamide in the mixture: 25 ꢀM). The
mixture was reacted for 6 h at 90 ºC, and then taken to dryness. HPLC analysis of the
crudes (dissolved in water) showed complete deprotection of maleimides and high to
quantitative conjugation yields.
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This procedure was found to be a good alternative to accelerate cycloaddition reactions
not completed even after days reaction time (preparation of conjugate 27, see
Supporting Information).
RESULTS AND DISCUSSION
Synthesis of [protected maleimido]-containing monomers.
For the synthesis of the lysine derivative 3 (Scheme 2a), the carboxyl group of 126 was
activated to give N-hydroxysuccinimido ester 2, and subsequent reaction of 2 with the
ε-amine of Fmoc-L-lysine linked the protected maleimide to the amino acid side chain
and gave the target derivative 3.
The PNA monomer 5 (Scheme 2b) was prepared by first reacting 1 with the secondary
amine of N-(N-Fmoc-2-aminoethyl)glycine tert-butyl ester, which was followed by an
acid treatment to deprotect the carboxyl group of 4.
Scheme 2c summarizes the reactions carried out to obtain [protected maleimido]-amine
9. In the preliminary steps, 2,5-dimethylfuran-protected maleimide (6, mixture of exo
and endo isomers) and N-Boc-aminoethanol (7) were prepared. Then a DIAD/Ph3P-
mediated Mitsunobu reaction between 6 and 7, followed by reaction with a 2:3
MeOH/conc. aq. ammonia mixture to get rid of the endo isomer26 afforded the fully
protected maleimido-amine 8. Finally, removal of the Boc group by treatment with
trifluoroacetic acid furnished amine 9 (trifluoroacetate salt).
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These [protected maleimido]-containing monomers were used in the solid-phase
synthesis of peptide, PNA and peptoid polyamides following well-known procedures
(see Supporting Information for experimental conditions, and Schemes S1-S3).
Syntheses proceeded smoothly, showing that all derivatives behaved as standard
building blocks.
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Maleimide deprotection and conjugation reactions with polyamides.
Conditions for maleimide deprotection (retro–Diels-Alder reaction) were examined for
the different polyamides. Peptide 10, PNA 14 and peptoid 18 (Scheme 3) were heated
either in a metal block (suspension in toluene) or in a microwave oven (MeOH/H2O
solution), and the resulting crudes were analyzed by HPLC.
Peptide 10 afforded fairly heterogeneous crudes. Best results were achieved by heating
in toluene (Scheme 3a, a.1; Figure S2), but the yield was not very good, and the longer
the reaction time the lower the quality of the crude.
In the case of the PNA chain (14, Scheme 3b, b.1), highly homogeneous maleimido-
PNA crudes (15) were obtained with any of the two deprotection procedures, the yield
being higher when using the microwave oven (>95 %, Figure S6) than when heating in
toluene (ca. 70 %).
As to peptoid 18 (Scheme 3c, c.1), microwave irradiation did not afford a sufficiently
satisfactory crude. Yet, heating in toluene provided the target maleimido-peptoid (19) in
good yield (>90 %) and high purity (Figure S9).
Reaction (Scheme 3) of the maleimido-containing polyamides (peptide 11, PNA 15 and
peptoid 19) with thiol- and diene-containing compounds (Figure 1) afforded the target
conjugation products (12, 16, 17, and 20-23; Figures S3b, S7 and S10) and confirmed
that maleimide deprotection had afforded fully reactive maleimides in all cases.
Scheme 3, Figure 1
With the aim to improve the low yields associated with maleimide deprotection in
peptides, we considered the possibility of carrying out one-pot deprotection and
conjugation reactions, in other words, of deprotecting the maleimide in the presence of
either the thiol or the diene. Previously, the stability of the adducts resulting from
conjugation reactions (Michael thiol-ene and Diels-Alder) to maleimide deprotection
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conditions was verified using standard samples of conjugates 12 and 13 (see
Supplementary Information, section 4). Both 12 and 13 remained undegraded, which
indicated that maleimide deprotection conditions are compatible with maleimide-
involving conjugation reactions (Figure S13).
9
When peptide 10 was suspended in toluene and heated in the presence of glutathione
over a period of 6 h (Scheme 3a, a.3), the target conjugate 12 was obtained in high yield
and purity (Figure S3b). Likewise, conjugate 13 was the main product in the crude
(Figure S4) when 10 and diene-dT5 were reacted under the same conditions (Scheme 3a,
a.4). These results suggest that the thiol or the diene present in the reaction medium
drive the equilibrium of the retro–Diels-Alder reaction by trapping the free maleimide,
and that the conjugation reaction is quick enough so as to prevent the maleimide from
being degraded. Therefore, when the two reactions are simultaneously carried out the
overall yield is higher, and the final conjugate is much more homogeneous. This result
has also been exploited for the preparation of cyclic oligonucleotides.30
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Both the Michael-type and Diels-Alder reactions provide conjugates containing
succinimide rings (see structures in Schemes 1 and 3). These succinimides were fairly
stable in aqueous media, with hydrolysis taking place only after several cycles of
dissolving in water and lyophilization, except in the case of conjugate 21. 21 Could only
be obtained if 19 was reacted with an excess (10 equiv) of biotin-SH for 5 min, after
which time the mixture had to be immediately frozen and lyophilized. Longer reaction
times gave crudes in which the main product was the conjugate with the hydrolyzed
succinimide. Conjugate 28 (see Scheme 4 and below) also underwent some hydrolysis
(37 %) upon microwave-promoted maleimide deprotection.
Two maleimide-involving consecutive click reactions were carried out on peptide 24,
PNA 31, and a PNA sequence described to exhibit antibactericidal antisense effects31
(39) (Scheme 4).
Scheme 4
Synthesis of conjugates differing in the position to which glutathione and diene-dT5
were linked to peptide 24 (Scheme 4a, 1.i and 2.i, Figures S15 and S16) involved,
firstly, reactions with the N-terminal free maleimide, which yielded conjugates 25 and
28. 25 And 28 were then heated in toluene to deprotect the internal maleimides,
providing compounds 26 and 29. Finally, reaction of 26 and 29 with diene-dT5 and
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glutathione, respectively, afforded the doubly-derivatized regioisiomeric conjugates 27
and 30.
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9
As in previous experiments, the maleimide deprotection yield was lower for the peptide-
peptide conjugate (25) than for the peptide-oligonucleotide one (28). Yet, carrying out
the deprotection and conjugation reactions simultaneously (Scheme 4a, 3.ii)
significantly ameliorated (Figure S15d) both the deprotection and the conjugation yield
(the room temperature cycloaddition providing 27 from 26 was extremely slow). Hence,
and in agreement with the outcome of the experiments described above, the yield of
maleimide deprotection in the presence of a maleimide-trapping compound was higher
than in its absence. It is also worth noticing that maleimide deprotection (retro–Diels-
Alder reaction) and Diels-Alder conjugation can be simultaneously carried out because
of the higher stability of diene-maleimide cycloadducts with respect to 2,5-
dimethylfuran-maleimide adducts.
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As to PNA 31, the final conjugates resulted from combining either two Michael-type
reactions, or, again, Michael and Diels-Alder reactions in either order (Scheme 4b). In
the first case, conjugate 34 incorporated biotin and a 21-aminoacid peptide (Cys-
peptide, Figure 1). In conjugate 35 the PNA scaffold was attached to biotin and a 10-
mer phosphorothioate oligonucleotide, while in 38 the 10-mer phosphorothioate
oligonucleotide and the Cys-peptide were linked to the PNA chain (Figures S18 and
S19).
Finally, the two ends of PNA 39 were successfully modified (Scheme 4c) using the
same three-step conjugation/maleimide deprotection/conjugation procedure (Figure
S20). Hence, this methodology is compatible with PNAs containing all of the four
nucleobases, and allows PNAs to be derivatized and keep their full recognition
potential.
The fact that no side reactions were detected during the preparation of conjugates 35, 38
and 43 further supports our previous conclusions on reactions involving thiophosphate
diesters and maleimides.27 Both HPLC and mass spectrometric analyses (see
Supplementary Information) are consistent with: i) phosphorothioate diesters not
interfering with maleimide-diene Diels-Alder cycloadditions (reactions affording 35 and
43), ii) microwave-promoted maleimide deprotection of the [phosphorothioate
oligonucleotide]-PNA conjugate cleanly affording the target compound (37), and iii)
successful reaction of the resulting free maleimide with the Cys-peptide (to yield 38).
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CONCLUSIONS
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2,5-Dimethylfuran-protected maleimide moieties suitable for use in the synthesis of
[protected maleimido]-containing peptides, PNAs and peptoids were prepared and
incorporated into the corresponding oligomers.
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Best conditions for maleimide deprotection were found to vary depending on the
oligomer, but in all cases fully reactive maleimides were obtained. Deprotection of
peptide-linked maleimides was troublesome, since the reaction took place in low yield
and was less clean than with PNAs and peptoids. Yet, these problems could be
overcome by carrying out simultaneously maleimide deprotection and conjugation,
since the adducts generated from Michael-type and Diels-Alder reactions remain stable
under maleimide deprotection conditions. One-pot maleimide deprotection and
conjugation furnished crudes substantially more homogeneous, increased the
conjugation yield, and accelerated slow cycloadditions.
Finally, peptides and PNAs incorporating the new [protected maleimido]-containing
derivatives and an unprotected maleimide at the N-terminal were employed as scaffolds
to which two different units were appended. This dual conjugation was accomplished
using either a three step conjugation/maleimide deprotection/conjugation procedure, or
the two-step conjugation/[maleimide deprotection + conjugation] alternative process.
The methodology here described broadens the scope of possibilities available so far for
the ligation of multiple components, the synthesis of complex molecular systems and
libraries of differently decorated scaffolds, or to attach different labels or reporter
groups to biomolecules (or their ligands). Decoration of a scaffold with different
appendices is made possible simply by the introduction of two maleimides, one free and
one protected. Moreover, incorporation of other functional groups can also be envisaged
to allow for further derivatization, since the thiol-maleimide and the Diels-Alder
reactions can be combined with other conjugation chemistries.
ACKNOWLEDGMENTS
This work was supported by funds from the Ministerio de Economía y Competitividad
(grant CTQ2010-21567-C02-01, and the project RNAREG, grant CSD2009-00080,
funded under the programme CONSOLIDER INGENIO 2010), and the Generalitat de
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Catalunya (2009SGR-208). A. S. and X. E. were recipient fellows of the Generalitat de
Catalunya and the MINECO, respectively.
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Supporting Information Available: General materials and methods; details on the
synthesis of the different compounds and mass spectrometric characterization;
assessment of the stability of Michael-type adducts and Diels-Alder cycloadducts to
maleimide deprotection conditions; HPLC traces of crude maleimido-polyamides and
derived conjugates. This information is available free of charge via the Internet at
http://pubs.acs.org/.
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E. (2010) Oligonucleotide carbohydrate-centered galactosyl cluster conjugates
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(26) Sánchez, A., Pedroso, E., and Grandas, A. (2011) Maleimide-dimethylfuran exo
adducts: effective maleimide protection in the synthesis of oligonucleotide conjugates.
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(27) Sánchez, A., Pedroso, E., and Grandas, A. (2012) Conjugation reactions involving
maleimides and phosphorothioate oligonucleotides. Bioconjugate Chem. 23, 300-307.
(28) Zuckermann, R. N., Kerr, J. M., Kent, S. B. H., and Moos, W. H. (1992) Efficient
method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer
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Boc protection under solvent- and catalyst-free conditions. Synlett 5, 806-808.
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(31) Good, L., Awasthi, S. K., Dryselius, R., Larsson, O., and and Nielsen, P. E. (2001)
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Bactericidal antisense effects of peptide-PNA conjugates. Nat. Biotechnol. 19, 360-364.
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FIGURES AND SCHEMES
Scheme 1. a) General procedure for polyamide synthesis and conjugation. b) General
structures of the conjugates prepared making use of two successive click reactions
(peptide and PNA sequences are shown in Scheme 4; the structure of thiols and dienes
is shown in Figure 1).
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Scheme 2. Synthesis of compounds suitable for the introduction of protected
maleimides into peptides (a), PNAs (b) and peptoids (c).
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Figure 1. Structures of thiols and dienes used in conjugation reactions.
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Bioconjugate Chemistry
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Scheme 3. Structures of peptide (a), PNA (b) and peptoid (c) incorporating a protected
maleimide unit, and reactions subsequently carried out.
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Scheme 4. Structures of peptide (a) and PNA scaffolds (b, c) incorporating two
maleimides, one protected and one unprotected, used for the attachment of two different
moieties, and subsequent deprotection and conjugation reactions.
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