Highly efficient synthesis of peptide-oligonucleotide conjugates: Chemoselective oxime and thiazolidine formation

Article abstract of DOI:10.1002/1521-3765(20010917)7:18<3976::AID-CHEM3976>3.0.CO;2-X

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an ox

Full text of DOI:10.1002/1521-3765(20010917)7:18<3976::AID-CHEM3976>3.0.CO;2-X

FULL PAPER
Highly Efficient Synthesis of Peptide ± Oligonucleotide Conjugates:
Chemoselective Oxime and Thiazolidine Formation**
Damien Forget, Didier Boturyn, Eric Defrancq,* Jean Lhomme, and Pascal Dumy[a]
Abstract: A convergent strategy for the
synthesis of peptide ± oligonucleotide
conjugates (POC) is presented. Chemo-
selective ligation of peptide to oligonu-
cleotide was accomplished by oxime and
thiazolidine formation. Oxime conjuga-
tion was performed by treating an oxy-
amine-containing peptide with an alde-
hyde-containing oligonucleotide or vice
versa. Ligation by thiazolidine forma-
tion was achieved by coupling a peptide,
acylated with a cysteine residue, to an
oligonucleotide that was derivatised by
an aldehyde function. For both ap-
proaches, the conjugates were obtained
in good yield without the need for a
protection strategy and under mild
aqueous conditions. Moreover, the ox-
ime ligation proved useful for directly
conjugating duplex oligonucleotides.
Combined with molecular biology tools,
this methodology opens up new pros-
pects for post-functionalisation of high-
molecular-weight DNA structures.
Keywords: chemoselectivity ´ con-
jugation
peptides
´
oligonucleotides
´
enhancement of cellular uptake, improvement of nuclear
localisation or an increase in affinity for the DNA or RNA
target.[3]
Introduction
The use of oligonucleotides (ODN) for the inhibition of gene
expression represents an attractive therapeutic approach.
Oligonucleotides can target mRNA (antisense strategy),
double stranded DNA (triple-helix strategy) or proteins
(aptamer strategy). However, a number of hurdles have to
be overcome. Cell-specific delivery, cellular uptake efficiency,
stability against nucleases, intracellular distribution and target
affinity constitute the main properties required for a ther-
apeutic oligonucleotide approach.[1] A number of chemically
modified oligonucleotides have been prepared for these
purposes. These include modification of the backbone (i.e.,
phosphorothioate analogues, PNA...), modification of the
sugar (i.e., 2'-O-alkylated sugar, a-nucleoside...) and attach-
ment of a variety of reporter groups at the 3' or 5'
extremities.[2]
Different methods for the preparation of peptide ± oligo-
nucleotide conjugates have been described.[3] Some have been
prepared successfully by stepwise solid-phase synthesis.
However, the standard chemistry required for peptide and
DNA synthesis is not fully compatible; this greatly limits the
scope of this approach.[3, 4] Therefore, the fragment-coupling
approach has emerged as a more general, suitable strategy. It
involves separate preparation of the peptide and of the
oligonucleotide, and subsequent coupling of the two purified
moieties. Various linkages, such as disulfide,[5] maleimide,[6]
thioether[7] and amide[8] have been used for this purpose.
Although many methods of conjugation have been success-
fully exploited, they still present drawbacks that restrict their
general applications. For instance, the major problem in using
thiol- or amine-based chemistry for conjugation is the lack of
regiospecific ligation upon reaction with peptides that contain
multiple cysteine or lysine residues. To circumvent these
limitations, the development of more selective and efficient
reactions, as well as easier procedures and work up, is still of
great interest. Ideally, the conjugation should take place
between fully unprotected peptides and oligonucleotides, with
minimal chemical manipulation and under physiological
conditions, thus allowing single-step synthesis of various
conjugates. Considering these criteria, we investigated the
use of two new linkages: the oxime linkage and the
thiazolidine linkage. Both have been used efficiently for the
chemical ligation of peptides[9] and for the conjugation of
peptides with carbohydrates.[10] The high efficiency and
In this context, peptide ± oligonucleotide conjugates (POC)
have received considerable attention as they can deliver a
large spectrum of properties to oligonucleotides, such as
[a] Dr. E. Defrancq, D. Forget, Dr. D. Boturyn,
Prof. J. Lhomme, Prof. P. Dumy
Â
LEDSS, UMR CNRS 5616, Universite Joseph Fourier
BP 53, 38041 Grenoble Cedex 9 (France)
Fax : (‡33)4-76-51-43-82
E-mail: Eric.Defrancq@ujf-grenoble.fr
[**] Abbreviations: Aloc ˆ allyloxycarbonyl, Boc ˆ t-butyloxycarbonyl,
DIEA ˆ diisopropylethylamine, EDTˆ ethanedithiol, Fmoc ˆ 9-fluo-
renylmethyloxycarbonyl,
MBHA ˆ p-methylbenzylhydrylamine,
Pmc ˆ 2,2,5,7,8-pentamethylchroman-6-sulfonyl, PyBOP ˆ benzotri-
azol-1-yl-oxy-tris-pyrolidino-phosphoniumhexafluoro
phosphate,
TIS ˆ triisopropylsilane, Trt ˆ trityl.
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3976±3984
selectivity of the aminooxy ± aldehyde coupling reaction has
been demonstrated by using this reaction for the determi-
nation of very low amounts of carbonyl derivatives in water
and ice,[11] as well for the quantification of abasic sites in
DNA.[12] This methodology has been extended to the labelling
of oligonucleotides and RNA,[13] and to the anchoring of
oligonucleotides on polymer supports.[14]
In this paper, the preparation of peptide ± oligonucleotide
conjugates by oxime bond formation (i.e., reaction between
an aldehyde and an oxyamine) and thiazolidine formation
(i.e., reaction between an aldehyde and a 1,2-aminothiol) is
described (Scheme 1). For this purpose, an aldehyde and an
Results and Discussion
Oxime-bond formation was achieved by inserting the com-
plementary reactive nucleophilic or electrophilic moieties
into both modules for comparison. On the one hand, the
aldehyde and the aminooxy group were introduced at the 5'-
extremity of the oligonucleotides by using phosphoramidites 1
and 2, respectively (Schemes 2 and 3, below). On the other
hand, the N-terminal position or the lysine side chain of the
peptides was functionalised by an aldehyde and an aminooxy
moiety through NaIO4 oxidative cleavage of a serine residue
and by using the N-Boc-O-(carboxymethyl)hydroxylamine
15, respectively (Schemes 4 and 5, below). For thiazolidine
formation, the peptide was derivatised with a cysteine at the
N-terminal position or at the lysine side chain, with the
oligonucleotide bearing the aldehyde function at the 5'-end.
Functionnalisation at the 5'-extremity of the oligonucleotides:
Aldehyde-containing linker: Preparation of oligonucleotides
containing an aldehyde linker at the 5'-extremity has already
been described.[20] The authors used a phosphoramidite linker
bearing a bis-benzoyl-protected diol. After DNA synthesis,
the aldehyde was generated by oxidative cleavage of the diol
with NaIO4. However, five steps were necessary for the
preparation of the corresponding phosphoramidite. We there-
fore considered a more straightforward synthetic route for the
preparation of the phosphoramidite. As depicted in Scheme 2,
phosphoramidite 1 was prepared in a two-step reaction from
commercial 1,2,6-hexanetriol. The 1,2-diol moiety was first
protected as a 2-methoxybenzylidene acetal by using 2-meth-
oxybenzaldehyde dimethylacetal in the presence of a catalytic
amount of pyridinium paratoluene sulfonate to afford the
protected diol 3.[21a] We noted that protection by a 2,4-
dimethoxybenzylidene acetal was unsuitable for automated
oligonucleotide synthesis due to its instability. Phosphityla-
tion of 3 with 2-cyanoethyl diisopropylchlorophosphoramidite
afforded the phosphoramidite 1 in 62% overall yield.
Scheme 1. Strategy for POCs synthesis by using oxime and thiazolidine
linkages.
aminooxy function were incorporated into the oligonucleo-
tide module at the 5'-extremity, while the peptide module
carried the corresponding complementary function; that is,
the aminooxy or the 1,2-aminothiol for reaction with the
aldehyde oligonucleotide, or the a-N-glyoxylyl for reaction
with the aminooxy-containing oligonucleotide (Scheme 1).
The interest of this new approach is emphasised by using two
biologically relevant peptides: a) a cyclopentapeptide con-
taining an arginine-glycine-aspartic acid tripeptide motif
(RGD), which is known to be a powerful and selective ligand
of the avb3 integrin receptor.[15]
Such peptides have been stud-
ied for tumour targeting[16] as
well as for DNA delivery;[17]
and b) a nuclear localisation
signal sequence (NLS): the ba-
sic
peptide
APKKKRKV
which is derived from the sim-
ian virus 40 antigen. The syn-
thesis of oligonucleotides carry-
ing this NLS sequence has been
recently described, and the hy-
bridisation properties of the
conjugates with the comple-
mentary strand have been stud-
ied.[18] A cytomegalovirus luci-
ferase-NLS gene containing
this sequence has also been
recently prepared for transfec-
tion into cells.[19]
Scheme 2. Preparation of aldehyde-containing oligonucleotides 6a and 6b. a) 4-methoxybenzaldehyde dimethyl
acetal, PPTS; b) 2-cyanoethyldiisopropylchlorophosphoramidite, DIEA; c) automated oligonucleotide synthesis,
then 80% aqueous AcOH; d) NaIO4.
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E. Defrancq et al.
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Phosphoramidite 1 was used to prepare the oligonucleo-
tides 6a and 6b as illustrative examples. Coupling of 1 at the
last step of the automated oligonucleotide synthesis was
achieved in excellent yield. After deprotection of the nucleo-
bases under the usual conditions (ammonia treatment for 24 h
at 558C), the oligonucleotides 4a and 4b were purified by
reverse-phase HPLC. The 2-methoxybenzylidene acetal pro-
tection allowed easy purification of the oligonucleotides at
this stage due to the groupꢁs hydrophobic properties (Fig-
ure 1A). The 2-methoxybenzylidene acetal was then cleaved
under mild acidic conditions. Treatment of the 5'-end-
protected oligonucleotides 4a and 4b with 80% aqueous
acetic acid for 1 h, conditions that are commonly used for
dimethoxytrityl (DMT) deprotection, afforded the corre-
sponding oligonucleotides 5a and 5b. The methoxybenzyl-
idene group was found to be remarkably labile under these
conditionsÐa longer time is generally required for hydrolysis
of methoxybenzylidene derivatives.[21b] No side reaction was
observed. Compounds 5a and 5b were characterised by
electrospray ionisation mass spectrometry (ES-MS, Table 1).
Oxidative cleavage of the diol was then performed by using a
50-fold excess of NaIO4 at room temperature for 30 min; this
led to selective formation of the desired aldehydes 6a and 6b.
Table 1. ES-MS analysis.[a]
Compound
Calcd Mass
Found Mass
5a
5b
10
12
14
2566.8
3466.3
3708.7
3506.6
926.1
2568.2
3466.2
3710.9
3505.7
926.3
16
676.7
677.2
17
677.7
678.4
18
706.8
707.3
19
20
21
22
23a
23b
24
25
26
1271.5
1285.5
1254.4
1301.6
3193.4
4093.0
4687.8
4107.2
4702.0
3223.5
4123.1
4717.9
1271.4
1285.4
1254.2
1301.4
3192.5
4092.7
4689.0
4106.6
4698.7
3223.6
4123.9
4717.8
27a
27b
28
[a] Negative mode for oligonucleotides and conjugates, and positive mode
for peptides. Eluent: H2O/CH3CN 50:50 (v/v); flow rate: 8 mLmin 1. The
oligonucleotides and the conjugates were dissolved in H2O/CH3CN/NEt3
50:50:2 (v/v/v). The peptides were dissolved in H2O/CH3CN/TFA 50:50:1
(v/v/v).
Aminooxy-containing oligonucleotides: In a recent paper, we
described a convenient synthetic route to aminooxy-contain-
ing oligonucleotides by incorporating a trityl-protected ami-
nooxy group by using the phosphoramidite 2 as depicted in
Scheme 3.[22] The oxyamine 8 was obtained from reaction of
6-bromohexanol with the dicarboximide derivative, followed
by cleavage of the protecting group with hydrazine. Selective
N tritylation and subsequent phosphitylation with 2-cya-
noethyl tetraisopropylphosphorodiamidite gave the phos-
Figure 1. HPLC profiles (detection at 260 nm): A) crude protected
undecamer diol 4b; B) purified undecamer diol 5b; C) crude reaction
mixture of aldehyde-containing undecamer 6b with RGD peptide 16;
D) crude reaction mixture of aldehyde-containing undecamer 6b with NLS
peptide 19. For the HPLC conditions, see Experimental Section.
Scheme 3. Preparation of oxyamine-containing oligonucleotide 11. a) K2CO3, DMF; b) N2H4, EtOH; c) TrCl, pyridine; d) N,N'-diisopropylammonium
tetrazolide, 2-cyanoethyldiisopropylphosphorodiamidite, e) automated oligonucleotide synthesis, then 80% aqueous AcOH; f) acetone, H2O.
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Peptide ± Oligonucleotide Conjugates
3976±3984
phoramidite synthon 2. It was noted that N protection with
monomethoxytrityl chloride gave the corresponding amino-
oxy derivative, which proved highly unstable.
The undecamer d(XCGCACACACGC) 10, in which X
represents the 5'-aminooxylinker, was then prepared accord-
ing to standard b-cyanoethylphosphoramidite chemistry by
incorporating phosphoramidite 2 at the final step of the
automated synthesis. After deprotection with concentrated
ammonia for 24 h at 558C, the oligonucleotide 10 was purified
by reverse-phase HPLC (Figure 2A shows the crude protect-
Figure 2. HPLC profiles (detection at 260 nm): A) crude protected amino-
oxy undecamer 10; B) purified aminooxy undecamer 11; C) crude reaction
mixture of aminooxy undecamer 11 with RGD peptide 17; D) purified
peptide 25. For the HPLC conditions, see Experimental Section.
Scheme 4. Preparation of functionalised RGD peptides 16, 17 and 18.
a) 1%TFA, CH2Cl2; b) PyBOP, DIEA, high dilution, then tetrakis(triphe-
nylphosphine)Pd(0); c) 15, then 50% TFA; d) Boc-Ser(OtBu)-OH, Py-
BOP, then 50% TFA/TIS; e) NaIO4; f) Fmoc-Cys(Trt)-OH, PyBOP, then
piperidine/DMF, then 90% TFA/EDT/TIS.
ed aminooxy undecamer 10) and characterised by ES-MS
(Table 1). Removal of the trityl protection was then per-
formed in 80% aqueous AcOH at room temperature for 8 h
to afford the free aminooxy oligonucleotide 11 (Figure 2B).
We noticed that compound 11 reacts readily with even traces
of carbonyl compounds. Therefore, it was best characterised
by the adduct 12 formed immediately after addition of
acetone (ES-MS, Table 1).
coupling reagent. The Aloc moiety was then cleaved by using
tetrakis(triphenylphosphine)palladium(0) in the presence of
phenylsilane as scavenger.[24] Purification by reverse-phase
HPLC yielded the key intermediate 14. Introduction of the
aminooxy group was achieved by coupling the activated ester
of N-Boc-O-(carboxymethyl)-hydroxylamine, 15, onto the
lysine side chain. First, the aminooxy moiety was protected by
a Boc group from commercial carboxymethoxylamine hemi-
hydrochloride to give the acid 15.[25] Before the coupling
reaction, the acid was reacted with N-hydroxysuccinimide in
the presence of dicyclohexyl carbodiimide to give the
corresponding activated ester. Reaction of the activated ester
of 15 with the peptide 14 was carried out in DMF. The
protecting groups were then removed in 50% trifluoroacetic
acid (TFA) in the presence of triisopropylsilane. The resulting
aminooxy peptide 16 was purified by HPLC. The serine
residue was introduced by using commercially available Boc-
Ser(OtBu)-OH and PyBOP, and then the protecting groups
were removed in 50% TFA. The 1,2-amino alcohol was then
oxidised by using NaIO4; this led to the selective formation of
the aldehyde-containing peptide 17. The cysteine residue was
Peptides synthesis:
RGD peptide: (Scheme 4) The protected cyclic peptide 14
represents the key intermediate into which the reactive
moiety (i.e., ONH2, CHO or SH) can be anchored through
the amino side-chain group of the lysine residue. The synthesis
of compound 14 has been reported.[23] It was prepared with
some modifications by cyclisation of the linear peptide
H-Asp(OtBu)-phe-Lys(Aloc)-Arg(Pmc)-Gly-OH 13.[23c] The
linear side-chain-protected peptide was assembled by solid-
phase peptide synthesis starting with a glycine residue at the C
terminus to prevent racemisation during the subsequent head-
to-tail cyclisation. The latter was carried out at high dilution
(0.5mm) in DMF to prevent dimerisation, with PyBOP as
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E. Defrancq et al.
FULL PAPER
introduced by using Fmoc-Cys(Trt)-OH and PyBOP. Remov-
al of the Fmoc protecting group by using piperidine in DMF
and subsequent cleavage of tBu and Pmc protecting groups in
90% TFA with ethanedithiol as scavenger afforded the
corresponding peptide 18. The three peptides 16, 17 and 18
were characterised by ES-MS (Table 1).
The reactive functions were introduced directly onto the
support at the N-terminal position by using the activated ester
of 15, Boc-Ser(OtBu)-OH or Fmoc-Cys(Trt)-OH, which led,
after deprotection and cleavage from the resin, to peptides 19,
20 and 22, respectively. The aldehyde-containing peptide 21
was obtained from oxidation of the intermediate amino
alcohol 20 with NaIO4. Peptides 19 ± 22 were checked for their
purity by HPLC and were characterised by ES-MS (Table 1).
NLS peptide (Scheme 5): The NLS-containing peptides were
assembled manually on a MBHA resin to provide the
C-terminal-amidated peptide by using a Fmoc/tBu strategy.
Conjugation reactions (Scheme 6):
Oxime linkage: We first studied the conjugation of the
aldehyde-containing oligonucleotides 6a and 6b with the
RGD-containing peptide 16. A slight excess (1.5 equiv) of the
aminooxy peptide 16 was reacted with the 8-mer 6a and the
11-mer 6b in aqueous solution at pH 5. Reactions were
carried out in slightly acidic conditions as the optimal pH is
around 4 ± 5 for oxime-bond formation.[26] The course of the
reaction was followed by reverse-phase HPLC, and the
reaction proceeded essentially to completion within 1 h to
yield exclusively the oxime 23b, as depicted in Figure 1C. The
same selectivity was observed for conjugation with 6a.
Subsequent purification by HPLC afforded the conjugates
23a and 23b in almost 50% isolated yield. The same protocol
was then applied to the NLS peptide 19. Reaction of the 11-
mer 6b with the aminooxy peptide 19 in aqueous solution
selectively afforded the conjugate 24 (Figure 1D). The con-
jugates 23a, 23b and 24 were characterised by ES-MS. In all
cases, the experimentally determined molecular weights were
in excellent agreement with the calculated values (Table 1).
The ªreverse strategyº was conducted by reacting the
oxyamine-containing oligonucleotide 11 and the aldehyde-
Scheme 5. Preparation of functionalised NLS peptides 19, 21 and 22. a) 15,
then 90% TFA/TIS; b) Boc-Ser(OtBu)-OH, PyBOP, then 90% TFA/TIS;
c) NaIO4; d) Fmoc-Cys(Trt)-OH, PyBOP, then piperidine/DMF, then 90%
TFA/EDT/TIS.
Scheme 6. Conjugation reactions: synthesis of POCs 23 ± 28.
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Peptide ± Oligonucleotide Conjugates
3976±3984
containing peptides 17 and 21. The reactions were performed
under the aforementioned conditions and led selectively to
the expected conjugates 25 (Figure 2C) and 26, respectively.
The conjugates 25 and 26 were purified by RP-HPLC and
characterised by ES-MS (Table 1). In both cases, the yields
were close to 50% after purification by HPLC.
bond, it was found that the thiazolidine bond is less stable at
pH 4. In fact, the conjugate 27a is up to 50% hydrolysed back
to the starting material after 72 h of incubation at pH 4 at
378C. However, 27a proved to be stable at higher pH value
(e.g., pH 7) as emphasised by the lack of hydrolysis or by
product formation under this condition.
The chemical stability of the oxime linkage was then
studied by incubating the conjugates 23b and 25 in a
phosphate buffer at pH 4 and pH 7. Surprisingly, no apparent
difference of stability was observed for both conjugates,
although it was anticipated that the glyoxylic oxime 25 would
be more stable than the classical oxime 23b. No significant
hydrolysis or degradation products (from depurination) were
observed even after 72 h of incubation at 378C.
Oxime-bond formation by using these two strategies
permits rapid and clean preparation of peptide ± oligonu-
cleotide conjugates. However, it should be emphasised that
the approach involving coupling of an aminooxy oligonucleo-
tide to an aldehyde peptide appeared less convenient to work
up. In fact, we noticed that free aminooxy oligonucleotides
are very often prone to react with traces of carbonyl
compounds present in HPLC solvents or in the atmosphere.
Therefore, the first approach is recommended for the syn-
thesis of oxime conjugates.
Hybridisation properties of the POC: The hybridisation
properties of the conjugates were studied by melting temper-
ature (Tm) measurements to evaluate the influence of the two
peptides RGD and NLS as well as of the two linkages on the
stability of the duplex oligonucleotide. The oxime conjugates
23b and 24 and the thiazolidine conjugates 27b and 28,
containing, respectively, the RGD and the NLS peptide, were
hybridised with their complementary strand d(GCGTGTG-
TGCG). The melting temperatures of the resulting duplexes
were determined (Table 2). The oligonucleotide 5b contain-
Table 2. Melting temperatures of duplexes formed by hybridisation of the
indicated oligonucleotides with the complementary strand d(GCGTGTG-
TGCG).[a]
Parent oligonucleotide 5b
POC 23b (oxime linkage)
POC 27b (thiazolidine linkage)
POC 24 (oxime linkage)
Tm ˆ 66.08C Æ 18C
Tm ˆ 68.08C Æ 18C
Tm ˆ 69.08C Æ 18C
Tm ˆ 66.58C Æ 18C
Tm ˆ 66.58C Æ 18C
Thiazolidine linkage: Formation of the thiazolidine linkage
was first performed with the RGD peptide 18 and the 8-mer
6a. The reaction was conducted at room temperature in
sodium acetate buffer at pH 5.4 with a fourfold excess of
peptide 18.[27] The reaction was very rapid (t1/2 < 15 min) and
led to selective formation of the conjugate 27a (Figure 3A).
POC 28 (thiazolidine linkage)
[a] Measurements were performed in phosphate buffer (sodium phosphate
buffer (10mm), EDTA (1mm), NaCl (100mm), pH 7).
ing the diol linker at the 5'-end was studied for comparison.
All the peptide ± oligonucleotide conjugates showed a slightly
higher melting temperature. The slight increase of stability
could be attributed to the positive charges of the lysine and
arginine side chains present within each peptide. Such
stabilisation of conjugates carrying basic amino acids has
been previously reported.[6a, 18] It is also notable that the
nature of the linkage does not appear to perturb the stability
of the double strand, as no substantial difference was
observed between 23b and 27b or between 24 and 28.
Conjugation with duplex oligonucleotides: The undecamer
6b was hybridised with the complementary strand, and the
duplex was reacted with the RGD peptide 16 under the same
conditions as for the single strand. The reaction was complete
within 2 h and showed the same efficiency as previously
observed when the single strand was used. The absence of
variation of the melting temperature during the course of
reaction clearly indicates that the coupling occurs without
disrupting the duplex structure.
Figure 3. HPLC profiles (detection at 260 nm): A) crude reaction mixture
of aldehyde-containing oligonucleotide 6b with RGD peptide 18; B) crude
reaction mixture of aldehyde-containing undecamer 6b with NLS peptide
22. For the HPLC conditions, see Experimental Section.
The same procedure was then applied to the 11-mer 6b with
the RGD peptide 18 and the NLS peptide 22. Exclusive
formation of conjugates 27b and 28 was observed, respec-
tively (Figure 3B shows the HPLC profile of the crude
reaction mixture in the case of conjugation with NLS peptide
22). The three conjugates 27a, 27b and 28 were characterised
by ES-MS (Table 1).
The chemical stability of the thiazolidine linkage was also
studied by incubating the conjugate 27a under the same
conditions as used for 23b and 25. In contrast to the oxime
Conclusion
Conjugation of oligonucleotides with peptides has been
readily performed through oxime or thiazolidine bond
formation. These one-step ligations are compatible with the
use of unprotected peptides and oligonucleotides. Thanks to
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(2CH), 114.1 (2CH), 104.3 and 103.41 (CH), 77.5 and 76.7 (CH), 71.1 and
70.4 (CH2), 63.0 (CH2), 55.7 (CH3), 33.6 (CH2), 32.9 (CH2), 22.4 (CH2); MS
(DCI/NH3): m/z ˆ 253 [M‡H] .
the high chemoselectivity of these reactions, the rapid and
convenient production of POCs without the need for exten-
sive chemical manipulation is thus allowed. The aldehyde-
and aminooxy-containing oligonucleotides are easily acces-
sible from phosphoramidites 1 and 2. Moreover, oxime and
thiazolidine ligations have already been carried out under
denaturing or high-salt conditions; this may extend the scope
of the present approach to less-soluble peptides or conjugates
as well as to higher molecular weight systems.[28] Together the
high efficiency and versatility of this strategy over conven-
tional conjugation methods is of great interest for devising
new molecular systems based on ODN. Furthermore, the
conjugation with oxime-bond formation could also be per-
formed on duplexesꢁ secondary structure. This latter result
opens up attractive prospects for the post-functionalisation of
high-molecular-weight structures, such as DNA, combined
with the molecular biology tools availability.
‡
Phosphoramidite (1): DIEA (375 mL, 2.10 mmol) and 2-cyanoethyl diiso-
propylchlorophosphoramidite (315 mL, 1.40 mmol) were added under
argon to a solution of compound 3 (270 mg, 1.10 mmol) in anhydrous
CH2Cl2 (3 mL). The solution was stirred for 2 h at room temperature, and
CH2Cl2 (20 mL) was then added. The organic layer was washed with 10%
aqueous NaHCO3 solution, then with brine and dried (Na2SO4). The
solvent was evaporated under vacuum, and the crude mixture was purified
by silica gel column chromatography (CH2Cl2/cyclohexane/Et3N 74:24:2)
1
to give compound 1 as a white oil. Yield: 0.40 g, 82%; H NMR (CDCl3):
d ˆ 7.37 (dd, 2H; Ar-H), 6.87 (d, 2H; Ar-H), 5.83 and 5.72 (2s, 1H; O-CH-
O), 4.24 ± 4.05 (m, 2H; CH2O), 3.78 (s, 3H; OCH3), 3.65 ± 3.55 (m, 5H; CH-
O and 2CH2O), 2.61 (m, 2H; CH2CN), 1.68 ± 1.52 (m, 8H; 3CH2 and 2CH),
1.18 ± 1.13 (m, 12H; 4CH3); 31P NMR (CDCl3): d ˆ 145.5; MS (FAB, NBA
‡
matrix): m/z ˆ 453 [M‡H] .
4-(6-Hydroxyhexyloxy)-4-aza-tricyclo [5.2.1.02,6]dec-8-ene-3,5-dione (7): A
mixture of endo-N-hydroxy-5-norbornene-2,3-dicarboximide (5.02 g,
28.0 mmol) and K2CO3 (7.70 g, 56.0 mmol) in DMF (250 mL) was stirred
at 508C under argon for 1 h. 6-Bromohexanol (5.07 g, 28.0 mmol) was
added, and the mixture was then stirred for 4 h at 508C. After filtration, the
solvent was evaporated under vacuum. EtOAc was added to the residue
obtained, and the organic layer was washed with 0.1n NaOH, then with
brine. The organic layer was dried (Na2SO4) and evaporated to give
compound 7 as a pale yellow oil. Yield: 6.00 g, 78%; 1H NMR (CDCl3): d ˆ
Experimental Section
Materials and methods: All commercially available chemical reagents were
used without purification. 2-Cyanoethyl diisopropylchlorophosphorami-
dite and 2-cyanoethyl tetraisopropylphosphorodiamidite were purchased
from Aldrich. 4-Methoxybenzaldehyde dimethyl acetal and the N-Boc-O-
(carboxymethyl)hydroxylamine, 15, were prepared as described.[21, 25]
Compound 15 is also commercially available from Fluka as (Boc-amino-
oxy)acetic acid. Thin-layer chromatography (TLC) was performed on silica
gel 60F254 plates (Merck), and preparative column chromatography was
performed on silica gel 60 (Merck, 200 ± 63 mm). HPLC purification, as well
as HPLC analysis of oligonucleotides and conjugates, was performed on a
Waters system equipped with two M510 pumps, an M490E detector and an
M680 system controller. HPLC purification and analysis of peptides were
done on a Waters system consisting of a Delta600 pump, a 2487 dual l
detector and a 600E-system controller. The oligonucleotides and the
conjugates were purified on a m-bondapak C-18 column (Macherey ± Nagel
Nucleosil: 10 Â 250 mm, 7 mm) with two systems of solvent. System I:
solvent A, 20mm ammonium acetate/CH3CN 95:5 (v:v); solvent B,
CH3CN; flow rate, 4 mLmin 1; a linear gradient from 0 to 30% B in
20 min was applied. System II: solvent A, 20mm sodium phosphate/MeOH
95:5 (v:v); solvent B, MeOH; flow rate, 4 mLmin 1; a linear gradient from
0 to 35% B in 20 min was applied. The peptides were purified on a Delta
PakTM C-18 column (Waters: 25 Â 200 mm, 15 mm) by using solvent system
III: solvent A, H2O/TFA 99.9:0.1 (v:v); solvent B, CH3CN/H2O/TFA
90:10:0.1 (v:v:v); flow rate 22 mLmin 1; a linear gradient from 5 to 100%
B in 30 min was applied. 1H and 13C NMR spectra were recorded on Bruker
AC200 and Avance spectrometers at 200 and 300 MHz, respectively. Mass
spectra were measured on a Delsi ± Nermag R10-10 for EI and DCI, and on
a VG Platform (Micromass) for ESI. Analysis was performed in the
negative mode for the oligonucleotides and the conjugates and in the
positive mode for the peptides. The eluent was 50% aqueous acetonitrile
and the flow rate was 8 mLmin 1. The oligonucleotides and the conjugates
were dissolved in 50% aqueous acetonitrile, and 1% of NEt3 was added.
The peptides were dissolved in 50% aqueous acetonitrile, and 1% of TFA
was added.
ˆ
6.10 (m, 2H; CH CH), 3.90 (t, 2H; CH2O), 3.60 (t, 2H; CH2O), 3.40 (m,
2H; 2CH-C ˆ O), 3.15 (m, 2H; 2CH), 1.80 ± 1.30 (m, 10H; 5CH2);
13C NMR (CDCl3): d ˆ 172.1 (q), 134.2 (CH), 76.8 (CH2), 61.8 (CH2), 51.0
(CH2), 44.4 (CH), 42.3 (CH), 32.1 (CH2), 27.6 (CH2), 25.0 and 24.9 (CH2);
‡
MS (EI): m/z ˆ 279 [M] .
6-Aminooxyhexan-1-ol (8): Compound 7 (6.00 g, 22.0 mmol) was dissolved
in EtOH (75 mL), and hydrazine (1.40 g, 44.0 mmol) was added. The
solution was refluxed for 2 h, then filtered and evaporated under vacuum.
Purification by silica gel column chromatography (EtOAc/MeOH 95:5) of
1
the crude mixture afforded 8 as a white oil. Yield: 2.60 g, 92%; H NMR
(CDCl3): d ˆ 3.59 ± 3.52 (m, 4H; 2CH2O), 1.54 ± 1.43 (m, 4H; 2CH2), 1.31 ±
1.25 (m, 4H; CH2CH2); 13C NMR (CDCl3): d ˆ 76.4 (CH2), 62.9 (CH2), 33.0
‡
(CH2), 28.7 (CH2), 26.2 (CH2), 26.0 (CH2); MS (DCI): m/z ˆ 134 [M‡H] .
6-(N-Tritylaminooxy)-hexan-1-ol (9): Trityl chloride (6.60 g, 24.0 mmol)
was added to a solution of compound 8 (2.60 g, 20.0 mmol) in dry pyridine
(50 mL) cooled to 08C. The solution was stirred at room temperature under
argon for 4 h. MeOH (3 mL) was then added dropwise, and the solvent was
evaporated under vacuum. The residue obtained was dissolved in EtOAc,
and the organic layer was washed with H2O, then with brine. The organic
layer was then dried (Na2SO4) and evaporated. The crude mixture was
purified by silica gel column chromatography (EtOAc/cyclohexane/NEt3
40:60:1) to give compound 9 as a white powder. Yield: 3.40 g, 45%;
1H NMR (CDCl3): d ˆ 7.34 ± 7.25 (m, 15H; Ar-H trityl), 6.23 (brs, 1H; NH),
3.66 (t, 2H; CH2O), 3.57 (m, 2H; CH2O), 1.47 ± 1.42 (m, 4H; 2CH2), 1.21 ±
1.16 (m, 4H; 2CH2); 13C NMR (CDCl3): d ˆ 144.5 (q), 129.1 (CH), 127.6
(CH), 126.8 (CH), 77.0 (q), 73.9 (CH2), 63.0 (CH2), 32.7 (CH2), 28.4 (CH2),
25.9 (CH2), 25.5 (CH2).
Phosphoramidite (2): N,N'-Diisopropylammonium tetrazolide (0.24 g,
1.7 mmol) and 2-cyanoethyl tetraisopropylphosphorodiamidite (1.00 g,
3.9 mmol) were added to a solution of compound 9 (1.00 g, 2.4 mmol) in
CH2Cl2 (20 mL). The solution was stirred under argon at room temperature
for 16 h, then diluted with CH2Cl2 (100 mL). The organic layer was washed
twice with H2O, then with brine and dried (Na2SO4). The solvent was
evaporated under vacuum and the residue obtained was purified by silica
gel column chromatography (EtOAc/cyclohexane/NEt3 30:70:1) to afford
phosphoramidite 2 as a white oil. Yield: 1.60 g, 60%; 1H NMR (CDCl3):
d ˆ 7.33 ± 7.26 (m, 15H; Ar-H trityl), 6.26 (s, 1H; NH), 3.82 (m, 2H; CH2O),
3.68 ± 3.58 (m, 4H; CH2O), 2.62 (t, 2H; CH2CN), 1.60 ± 1.44 (m, 5H; 2CH2
and CH), 1.21 ± 1.17 (m, 17H; 4CH3, 2CH2 and CH); 31P NMR (CDCl3):
4-[2-(4-Methoxy-phenyl)-[1,3]-dioxolan-4-yl]-butan-1-ol (3): 1,2,6-hexane-
triol (3.10 g, 23.0 mmol) and a catalytic amount of pyridinium p-toluene
sulfonate (0.1 g) were added to a solution of 4-methoxybenzaldehyde
dimethylacetal (2.10 g, 12.0 mmol) in DMF (20 mL). The solution was
stirred at 508C under argon for 20 h, then the solvent was evaporated under
vacuum. The oily residue was dissolved in CH2Cl2, and the organic layer
was washed successively with aqueous NaHSO3, aqueous NaHCO3 and
brine, and evaporated to give 3 as a white oil. Yield: 2.22 g, 76%; 1H NMR
(CDCl3): d ˆ 7.37 (dd, 2H; Ar-H), 6.87 (d, 2H; Ar-H), 5.84 and 5.73 (2s,
1H; O-CH-O), 4.24 ± 4.03 (m, 2H; CH2O), 3.78 (s, 3H; OCH3), 3.66 ± 3.60
(m, 3H; CH-O and CH2O), 1.68 ± 1.42 (m, 7H; 3CH2 and OH); 13C NMR
(CDCl3): d ˆ 160.8 and 160.7 (q), 130.9 and 130.3 (q), 128.4 and 128.2
‡
d ˆ 145.4; MS (FAB, NBA matrix): m/z ˆ 575 [M] .
Oligonucleotides synthesis: Automated DNA synthesis was carried out on
an Expedite DNA synthesiser (Perkin ± Elmer) by using standard b-
cyanoethyl nucleoside phosphoramidites chemistry on a 1mm scale. After
cleavage from the solid support and deprotection by treatment with
3982
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Chem. Eur. J. 2001, 7, No. 18

Peptide ± Oligonucleotide Conjugates
3976±3984
concentrated ammonia (30%) for 24 h at 558C, the oligonucleotides 4a, 4b
and 10 were purified by HPLC (system I).
intermediate aminoalcohol 20 with NaIO4 (30 equiv) in water for 3 h
afforded the aldehyde peptide 21, which was purified by HPLC.
The cysteine residue was introduced by using Fmoc-Cys(Trt)-OH (2 equiv)
and PyBOP (2 equiv). The Fmoc group was then removed by treatment
with a piperidine/DMF solution (1:4, v/v) for 10 min. Cleavage from the
resin and deprotection of the side-chain groups were then performed in
TFA/EDT/H2O/TIS (90:5:2.5:2.5) for 2 h; after purification by HPLC, this
gave the peptide 22.
Diol-containing oligonucleotides (5a) and (5b): The 5'-protected oligonu-
cleotides 4a and 4b were treated with an 80% AcOH aqueous solution for
1 h. The residue obtained after lyophilisation was dissolved in water, and
the aqueous layer was extensively washed with Et2O to remove the
methoxybenzylidene by-product. Subsequent lyophilisation afforded the
oligonucleotides 5a and 5b, which were characterised by ES-MS (Table 1).
Conjugation through oxime-ether linkage:
Aldehyde-containing oligonucleotides (6a) and (6b): NaIO4 (50 equiv,
3.5 mg) was added to a solution of oligonucleotide 5a (0.320 mmol) in water
(500 mL), and the solution was stirred at room temperature for 15 min. The
resulting oligonucleotide 6a was then purified by HPLC (system II); yield:
0.280 mmol, 86%. The undecamer 6b was prepared in the same manner;
yield: 78%.
From aldehyde oligonucleotides 6a and 6b: A solution of RGD peptide 16
(1.5 equiv) in water (100 mL) was added to a solution of oligonucleotide 6a
(21 OD) in water (500 mL, pH adjusted to 5 with AcOH). The mixture was
stirred at room temperature, and the progress of the reaction was followed
by HPLC. The starting material disappeared in 1 h. Purification by HPLC
with system II afforded the conjugate 23a in 68% yield (15 OD). The
conjugate 23b was obtained in the same manner from oligonucleotide 6b in
66% yield. Conjugation with NLS peptide 19 was achieved by using the
same protocol with the undecamer 6b and led to the conjugate 24 in 50%
yield.
Aminooxy oligonucleotide (11): The 5'-trityl-containing oligonucleotide 10
was treated with an 80% AcOH aqueous solution for 8 h. The solvent was
then evaporated by lyophilisation to give compound 11, which was used
without further purification for the next conjugation reaction. An aliquot of
the oligonucleotide 11 was incubated with acetone and the corresponding
oxime ether 12 was characterised by ES-MS (Table 1).
From aminooxy oligonucleotide 11: A solution of the RGD peptide 17
(0.34 mmol) in water (200 mL) was added to a solution of oligonucleotide 11
(20 OD, 0.17 mmol) in AcOH (500 mL). The mixture was stirred at room
temperature for 3 h. The crude mixture was then purified by HPLC by
using system I to give the conjugate 25 in 51% yield. Conjugation with the
NLS peptide 21 was achieved in the same manner and led to conjugate 26 in
50% yield.
Peptide synthesis: Solid-phase peptide synthesis was performed by using
Fmoc/tBu chemistry on a 348 W peptide synthesiser (Advanced ChemTech)
with SASRIN resin (loading: 0.5 mmolg 1, scale: 150 mg), in the case of
RGD peptides, or manually with MBHA resin, in the case of NLS peptides
1
(loading: 0.4 mmolg
,
scale: 150 mg). The coupling reactions were
performed for 30 min by using a twofold excess of amino acid protected
Conjugation by thiazolidine linkage:
by N-Fmoc and PyBOP and a fourfold excess of DIEA in DMF (1.5 mL).
The N-Fmoc protecting groups were removed by treatment with
a
Conjugation with RGD peptide 18: A solution of RGD peptide 18 (4 equiv)
in sodium acetate buffer (130 mL) was added to a solution of oligonucleo-
tide 6a (25 OD) in sodium acetate buffer (100 mL, pH 5.4). The mixture
was stirred at room temperature for 30 min until the starting material
disappeared. Purification by HPLC with system II afforded the conjugate
27a in 56% yield (14 OD). By using a similar protocol, reaction of the
oligonucleotide 6b gave the conjugate 27b in 50% yield.
piperidine/DMF solution (1:4 v/v, 10 mLg 1 resin) for 10 min, three times.
RGD peptides (16), (17) and (18): The key intermediate cyclic peptide 14
was obtained from the linear H-Asp(OtBu)-phe-Lys(Aloc)-Arg(Pmc)-Gly-
OH, 13. The linear peptide 13 was cleaved from the resin by reaction with
trifluoroacetic acid (1%) in CH2Cl2 for 20 min. The volatile solvent was
removed, and the resulting crude product was triturated, then washed with
Et2O three times and used without further purification. Cyclisation was
performed at 0.5mm concentration in DMF with PyBOP (1.2 equiv). The
pH was adjusted to 8 ± 9 by addition of DIEA, and the solution was stirred
at room temperature for 1 h. The Aloc moiety was then removed by using
tetrakis(triphenylphosphine)palladium(0) (0.2 equiv) and phenylsilane
(25 equiv). Purification by HPLC with system III gave the cyclic peptide
14 (77%). Introduction of the aminooxy moiety was achieved by reaction
of 14 with the N-succinimido-activated ester of the protected O-(carboxy-
methyl)hydroxylamine 15 (2 equiv) in DMF for 1 h in the presence of
DIEA. The protecting groups were then cleaved by treatment with 50%
TFA aqueous solution for 1 h; this led to the aminooxy-containing peptide
16 (68%). Introduction of the serine moiety was done by using Boc-
Ser(OtBu)-OH (2 equiv) and PyBOP (2 equiv) with DIEA (3 ± 4 equiv) in
DMF at 10 2 m. Cleavage of the protecting groups was then achieved by
treatment with TFA/H2O/TIS (95:2.5:2.5) solution for 2 h. Subsequent
oxidation was then performed by using NaIO4 (1.5 equiv) in water for 1 h.
Purification by HPLC afforded the aldehyde peptide 17 (30% overall
yield), which was characterised by ES-MS (Table 1).
Conjugation with NLS peptide 22: Conjugation with NLS peptide 22 was
achieved by using the same procedure as with the undecamer 6b and led to
the conjugate 28 in 64% yield.
Melting studies: The melting curves (absorbance versus temperature) were
measured at 260 nm on
a Lambda5 UV/visible spectrophotometer
equipped with a Perkin ± Elmer C570 ± 070 temperature controller and
1
by using a rate of 18Cmin (from 2 to 808C). Melting experiments were
carried out by mixing equimolar amounts of the two undecamer strands
dissolved in 10mm sodium phosphate buffer (pH 7) that contained 1mm
EDTA and 100mm NaCl. All measurements were done at a concentration
of 12mm. Before each melting experiment, samples were heated at 808C for
5 min, then cooled slowly.
Acknowledgement
This work was supported by the Association pour la Recherche sur le
Â
Ã
Cancer (ARC), the Region Rhone-Alpes and the Centre National de la
Recherche Scientifique (CNRS). The ªInstitut Universitaire de Franceº is
greatly acknowledged for financial support.
The cysteine residue was introduced by using Fmoc-Cys(Trt)-OH (2 equiv),
PyBOP (2 equiv) and DIEA (3 ± 4 equiv) in DMF at 10 2 m. The Fmoc
group was then cleaved by treatment with a piperidine/DMF solution (1:4,
v/v) for 10 min. The other protecting groups were then cleaved in TFA/
EDT/H2O/TIS (90:5:2.5:2.5) for 2 h; this led to peptide 18, which was
purified by HPLC (60%). The structure of 18 was confirmed by ES-MS
(Table 1).
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Chem. Eur. J. 2001, 7, No. 18
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0947-6539/01/0718-3983 $ 17.50+.50/0
3983

E. Defrancq et al.
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Received: March 1, 2001 [F3101]
3984
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Chem. Eur. J. 2001, 7, No. 18