NAA-modified dna oligonucleotides with zwitterionic backbones: Stereoselective synthesis of A-T phosphoramidite building blocks

Article abstract of DOI:10.3762/bjoc.11.8

Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive c

Full text of DOI:10.3762/bjoc.11.8

NAA-modified DNA oligonucleotides with zwitterionic
backbones: stereoselective synthesis of
A–T phosphoramidite building blocks
Boris Schmidtgall1,2, Claudia Höbartner3,4 and Christian Ducho*1,2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2015, 11, 50–60.
1Department of Chemistry, University of Paderborn, Warburger Str.
100, 33 098 Paderborn, Germany, 2Department of Pharmacy,
Pharmaceutical and Medicinal Chemistry, Saarland University,
Campus C2 3, 66 123 Saarbrücken, Germany, 3Max-Planck-Institute
for Biophysical Chemistry, Am Fassberg 11, 37 077 Göttingen,
Germany and 4Department of Chemistry, Institute of Organic and
Biomolecular Chemistry, Georg-August-University Göttingen,
Tammannstr. 2, 37 077 Göttingen, Germany
doi:10.3762/bjoc.11.8
Received: 24 October 2014
Accepted: 11 December 2014
Published: 13 January 2015
This article is part of the Thematic Series "Nucleic acid chemistry".
Guest Editor: H.-A. Wagenknecht
Email:
Christian Ducho* - christian.ducho@uni-saarland.de
© 2015 Schmidtgall et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
backbone modifications; DNA; nucleic acids; oligonucleotides;
stereoselective synthesis; zwitterions
Abstract
Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The
NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive
charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the
NAA-linkage at T–T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X–T motifs
(X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A–T phosphor-
amidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with
high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported
automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-
linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide
analogues.
Introduction
Oligonucleotides are important agents for a number of biomed- molecular recognition is a striking feature of nucleic acids, but
ical applications [1]. Thus, they are employed to exert antigene their high polarity represents a significant hurdle for cellular
[2] or antisense [3] mechanisms as well as to trigger or inhibit uptake and leads to problematic pharmcokinetics. Furthermore,
RNA interference [4]. The capability of sequence-specific they are prone to nuclease-mediated degradation. As a conse-
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Beilstein J. Org. Chem. 2015, 11, 50–60.
quence, it is of utmost importance to modify oligonucleotide such as 3 and 4 provides an 'NAA-modified oligonucleotide' 5,
structures using chemical or enzymatic methods in order to i.e., a nucleic acid strand with the NAA-linkage replacing a
develop oligonucleotide-based drug candidates or biomedical phosphate diester motif. The amino group of the NAA-modifi-
chemical probes [5,6].
cation is expected to display a positive charge at physiological
pH values, thus leading to a (partially) zwitterionic backbone
Native nucleic acids are connected by phosphate diesters as structure in NAA-modified oligonucleotides.
linking units, thus leading to a polyanionic backbone structure.
Implications of this characteristic feature of nucleic acid archi- We have previously described that NAA-modified DNA
tecture have been discussed by Westheimer [7] and Benner oligonucleotides can be obtained by standard solid phase-
[8,9], among others. However, the accumulation of negative supported automated DNA synthesis, using the 'dimeric' phos-
charges in the nucleic acid backbone is mainly responsible for phoramidite building blocks 6 (Figure 1) [38]. Overall,
their limited membrane penetration. Consequently, a signifi- 24 different oligonucleotide sequences with one to four NAA-
cant number of artificial internucleotide linkages has been modifications at various positions were synthesized. The stereo-
studied with the aim to manipulate the charge pattern in the chemistry of the NAA-motif was either (S) or (R) (obtained by
backbone and to enhance nuclease stability. The electroneutral application of the corresponding phosphoramidites (S)-6 or
nucleic acid mimic 'peptide nucleic acid' (PNA) [10-12] is (R)-6 for DNA synthesis) in order to study the influence of the
capable of sequence-specific binding to nucleic acids, but it was spatial orientation of the positive charge. Melting temperature
found to display limited water solubility and a peptide-like measurements showed that NAA-modified DNA oligonu-
folding behaviour [8]. For the selective replacement of some cleotides formed stable duplexes with native unmodified DNA
phosphate linkages in otherwise native oligonucleotide struc- or RNA counterstrands, although moderate destabilization in
tures, e.g., amide [13-20], triazole [21,22], phosphoramidate comparison to native duplexes was observed (particularly for
[23] and phosphate triester [24] moieties were reported along- DNA/RNA duplexes). Further experiments with native counter-
side a considerable number of other modifications.
strands bearing one nucleobase mismatch were performed, and
duplex structures were studied by CD spectroscopy. Overall, we
In comparison, the introduction of positive charges into the found that NAA-modified DNA oligonucleotides (i) formed
nucleic acid scaffold has found less attention. Positively stable duplexes with complementary counterstrands; (ii) were
charged units were attached to the 2'-hydroxy group or the fully capable of mismatch discrimination and (iii) formed
nucleobase as a compensation for the presence of negative duplexes without significant structural distortion, i.e.,
charges in the phosphate backbone [25-29]. In contrast to such B-form helices (DNA/DNA duplexes) and A-form helices
zwitterionic, but densely charged systems, only very few (DNA/RNA duplexes), respectively. It was concluded that
attempts were made to replace the phosphate moiety by a posi- typical chemical properties of nucleic acids are retained in
tively charged motif [30], mainly by Bruice et al. [31-34]. NAA-modified DNA oligonucleotides [38], thus making the
Selective replacement of some phosphates with cationic motifs NAA-linkage an interesting structural motif for oligonucleotide
furnished oligonucleotide 'chimera' [30] with zwitterionic back- analogues.
bone structures as reported both by Bruice [35,36] and
Letsinger [37].
Using 'dimeric' T–T phosphoramidites 6, it was only possible to
introduce the NAA-modification at T–T motifs. While this was
We have found the approach to prepare oligonucleotides with fully sufficient for initial studies, it will be of major importance
zwitterionic backbones interesting both for its fundamental to develop methods for the synthesis of NAA-modified oligonu-
implications and also for its potential contribution to the cleotides with the NAA-motif at more variable positions in the
existing 'toolbox' of backbone modifications for bioactive base sequence. The synthesis of 'dimeric' X–T phosphor-
oligonucleotide analogues. These considerations have led to our amidites (X = A, C, G) would enable an introduction of the
recently reported design of a novel artificial internucleotide NAA-linkage at every position in an oligonucleotide sequence
linkage named 'NAA-modification' (Figure 1) [38]. Ongoing with a T in 3'-direction, thus significantly broadening the ap-
synthetic and structure-activity relationship (SAR) studies on plicability of the modification. In this work, we describe the
naturally occurring muraymycin antibiotics (e.g., muraymycin stereoselective synthesis of 'dimeric' NAA-linked A–T phos-
A1 (1)) [39-46] have led to our previously reported synthesis of phoramidites (S)-7 and (R)-7 (Figure 1) as well as their applica-
'nucleosyl amino acid' structures 2 [47,48] as simplified tion in automated DNA synthesis. This represents the first step
5'-defunctionalized analogues of the muraymycin core motif. towards a comprehensive set of 'dimeric' NAA-linked X–T
Formally merging the nucleosyl amino acid (NAA) structure of phosphoramidites for the automated chemical synthesis of
type 2 with previously reported amide internucleotide linkages NAA-modified DNA oligonucleotides.
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Beilstein J. Org. Chem. 2015, 11, 50–60.
Figure 1: Design concept of nucleosyl amino acid (NAA)-modified oligonucleotides 5 formally derived from structures 1–4 (B1, B2 = nucleobases);
previously employed 'dimeric' T–T phosphoramidites 6 [38] for the automated synthesis of NAA-modified oligonucleotides; new 'dimeric' A–T phos-
phoramidites 7 as target structures of this study (DMTr = 4,4'-dimethoxytrityl).
Results and Discussion
3'-amino-2',3'-dideoxyadenosine 8 and the N-Fmoc-protected
For the synthesis of target phosphoramidites 7, it was planned thymidine-derived nucleosyl amino acids (S)-9 and (R)-9, res-
to employ a similar synthetic strategy as previously described pectively, via amide coupling, protecting group manipulation
by us for T–T phosphoramidites 6 [38]. One important objec- and phosphitylation. The stereoselective synthesis of both
tive was to construct the 6'-stereocenter of the NAA-linkage in a 6'-epimers of nucleosyl amino acid 9 has been reported before.
controlled fashion and to retain the resultant (6'S)- or the (6'R)- We have used 3-(N-BOM)-protected thymidine-5'-aldehyde 10
configuration, respectively, on the way to dimeric structures of (BOM = benzyloxymethyl), which can readily be obtained from
type 7 (Scheme 1). Thus, it was envisioned that target com- thymidine, in a sequence of Wittig–Horner reaction, asym-
pounds (S)-7 and (R)-7 could be obtained from protected metric hydrogenation of the resultant didehydro nucleosyl
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Beilstein J. Org. Chem. 2015, 11, 50–60.
Scheme 1: Retrosynthetic analysis of target phosphoramidites (S)-7 and (R)-7 (BOM = benzyloxymethyl).
amino acid and protecting group manipulations in order to provided satisfying results in the case of the corresponding
obtain both 6'-epimers of 9 dependent on the choice of the uridine derivatives [48]. The yield of the desired 5'-alcohols 15
hydrogenation catalyst [38]. Here, we report on further studies and 16 was limited though by partial concomitant cleavage of
directed towards a possible synthesis of 9 without 3-(N-BOM)- the 3'-O-TBDMS group upon prolonged reaction times. Alco-
protection of the thymine nucleobase. In the case of the corres- hols 15 and 16 were then oxidized to aldehydes 10 and 11 in
ponding uridine-derived nucleosyl amino acids, we have found quantitative yields using IBX in refluxing acetonitrile [50].
that uracil protection was not advantageous for the aforemen- With respect to their limited stability, thymidine-5'-aldehydes
tioned reaction sequence [48]. We have therefore decided to 10 and 11 were not stored, but directly used for the subsequent
employ both the 3-(N-BOM)-protected thymidine-5'-aldehyde Wittig–Horner reaction. They were therefore converted with
10 and also its thymine-unprotected congener 11 in glycine-derived phosphonate 12 [51-54] in the presence of
Wittig–Horner reactions with glycine-derived phosphonate 12 potassium tert-butoxide as a base. As anticipated [47,48,55],
and to compare both possible routes towards 9, i.e., with or these reactions showed pronounced stereoselectivity towards
without thymine protection (Scheme 1).
the Z-configured didehydro nucleosyl amino acids. In the case
of the reaction of 3-(N-BOM)-protected thymidine-5'-aldehyde
For the synthesis of the N-Fmoc-protected thymidine-derived 10 with phosphonate 12, isomer Z-17 was isolated in 71% yield,
nucleosyl amino acids (S)-9 and (R)-9, 3',5'-bis-O-silylated with E-17 representing a minor byproduct (3% yield) which
thymidine 13 (which can be readily prepared from thymidine could be separated by column chromatography. The assign-
with TBDMS chloride and imidazole in pyridine as solvent in ment of the configuration of the newly formed trisubstituted
quantitative yield) was 3-N-protected by alkylation with benzyl- C–C double bond was based on empirical 1H NMR criteria for
oxymethyl chloride (BOMCl), furnishing product 14 in 96% didehydro amino acids [56], which were proven to be useful
yield (Scheme 2). Although the route involving thymdine and reliable [38,47,48,57]. In the case of the conversion of 3-N-
protection has been published before [38], it is also depicted in unprotected thymidine-5'-aldehyde 11 with phosphonate 12, an
Scheme 2 in the interest of clarity and readability. Both bis-silyl unseparable mixture of Z-18 and E-18 was obtained in 66%
ethers 13 and 14 then underwent selective acidic cleavage in the overall yield (Z/E = 91:9). However, it is firmly established
5'-position using the conditions reported by Khan and Mondal that the subsequent asymmetric hydrogenation proceeds
[49], i.e., hydrochloric acid in methanol (generated by the reac- significantly faster with the Z-configured didehydro amino acid
tion of acetyl chloride with methanol), thus providing 3'-O- substrate [58], and it was therefore decided to use the
TBDMS-protected derivatives 15 and 16 in yields of 59% and aforementioned mixture of double bond isomers (containing 9%
66%, respectively. This method turned out to be advantageous of the unwanted E-isomer) as starting material for this transfor-
compared to 5'-O-desilylation mediated by TFA, which had mation.
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Beilstein J. Org. Chem. 2015, 11, 50–60.
Scheme 2: Synthesis of N-Fmoc-protected thymidine-derived nucleosyl amino acids (S)-9 and (R)-9; details on the reactions from 17 and 18 to 21
(asymmetric hydrogenation and subsequent protecting group manipulations) are given in Table 1.
Table 1: Reactions from 17 and 18 to 21 (see Scheme 2).
Asymmetric hydrogenation
Catalysta
Protecting group steps
#
Starting
material
Reaction
time
Yieldb
Additive
Yieldc
1
2
3
4
Z-17d
(S,S)-Me-DuPHOS-Rh
(R,R)-Me-DuPHOS-Rh
(S,S)-Me-DuPHOS-Rh
(R,R)-Me-DuPHOS-Rh
2 d
94% (S)-19
99% (R)-19
93% (S)-20
77% (R)-20
n-BuNH2
90% (S)-21
87% (R)-21
84% (S)-21
78% (R)-21
Z-17d
7 d
n-BuNH2
18 (mix.)e
18 (mix.)e
9 d
–
–
21 d
aHomogeneous chiral hydrogenation catalysts:
bd.r. >98:2 for all products; cover 2 steps from 19 or 20; dpure Z-isomer; eunseparable mixture of Z- and E-isomers (Z/E 91:9).
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Beilstein J. Org. Chem. 2015, 11, 50–60.
Asymmetric hydrogenation reactions were performed under be problematic particularly for reactions on a larger scale (>1 g
homogeneous conditions using the chiral catalysts (S,S)-Me- starting material). It is interesting to note that such limitations
DuPHOS-Rh or (R,R)-Me-DuPHOS-Rh, respectively [59]. It is were not encountered when the analogous nucleobase-unpro-
known that asymmetric hydrogenations of Z-configured dide- tected uridine-derived didehydro nucleosyl amino acid under-
hydro amino acids catalyzed by (S,S)-Me-DuPHOS-Rh give went asymmetric hydrogenation with the two aforementioned
L-amino acids and that analogous reactions catalyzed by (R,R)- catalysts [48]. This decreased reactivity might have been the
Me-DuPHOS-Rh provide D-amino acids [57,60]. This has also result of the presence of the E-isomer E-18 in the reaction mix-
been observed when these catalysts were applied for the syn- ture, which probably led to partial inhibition of the Rh(I) cata-
thesis of uridine-derived nucleosyl amino acids [47,48]. It was lyst.
therefore possible to direct the stereochemical outcome of the
hydrogenation reaction by the choice of either the (S,S)- or the In order to convert the hydrogenation products 19 and 20 into
(R,R)-catalyst. As reported previously [38], the hydrogenation the desired building blocks 9, three further transformations were
of pure 3-(N-BOM)-protected Z-17 in the presence of (S,S)-Me- necessary: (i) hydrogenolytic cleavage of the Cbz and, in the
DuPHOS-Rh thus furnished thymidine-derived nucleosyl amino case of 19, also of the BOM group; (ii) Fmoc-protection of the
acid (S)-19 in 94% yield, and its 6'-epimer (R)-19 was obtained 6'-amino functionality (furnishing intermediates (S)-21 and
from the same starting material in 99% yield using catalytic (R)-21) and (iii) cleavage of the tert-butyl ester (Scheme 2).
amounts of (R,R)-Me-DuPHOS-Rh (Table 1, entries 1 and 2). Using the two diastereomerically pure 6'-epimers of 3-(N-
Both transformations displayed excellent diastereoselectivies BOM)-protected 19 as starting material, one challenge was to
(d.r. >98:2 for both products), which indicated that these reac- avoid unwanted side reactions resulting from the generation of
tions proceeded in a catalyst-controlled fashion. The observa- formaldehyde in the reaction mixture. Hydrogenolysis of the
tion that full conversion of Z-17 to (S)-19 was reached after BOM group affords toluene and formaldehyde as byproducts,
2 days with the (S,S)-Me-DuPHOS-Rh catalyst, while comple- and the Cbz-deprotected 6'-amino group can undergo unwanted
tion of the reaction took 7 days with the (R,R)-Me-DuPHOS-Rh reductive amination, i.e., methylation, with the liberated
catalyst, suggested that the former transformation represented formaldehyde. Our method to prevent this side reaction was to
the apparent 'matched' case and the latter the apparent include an excess of n-butylamine as an additive in the reaction
'mismatched' case. This was also in agreement with similar mixture of the hydrogenolysis step. This way, the formalde-
findings for the synthesis of the according uridine-derived hyde methylated the added n-butylamine, furnishing a reason-
congeners [47,48]. When the isomeric mixture (Z/E = 91:9) of ably volatile byproduct [38,48]. Subsequent Fmoc-protection
the 3-N-unprotected didehydro amino acid 18 was employed as under standard conditions then afforded diastereomerically pure
starting material, 3-N-unprotected products (S)-20 (93% yield, products (S)-21 (90% yield over 2 steps from (S)-19) and (R)-21
with (S,S)-Me-DuPHOS-Rh) and (R)-20 (77% yield, with (R,R)- (87% yield over 2 steps from (R)-19), respectively (Table 1,
Me-DuPHOS-Rh) were obtained, again with excellent dia- entires 1 and 2). For the analogous transformation of 3-N-
stereoselectivities (d.r. >98:2 for both products, Table 1, entries unprotected (S)-20 and (R)-20, it was possible to omit the addi-
3 and 4). However, it was much more difficult to drive these tive n-butylamine, but yields were not improved due to this
reactions to completion as reflected by the significantly simplification (84% yield of (S)-21 over 2 steps from (S)-20,
prolonged reaction times (9 and 21 days, respectively). For the 78% yield of (R)-21 over 2 steps from (R)-20, Table 1, entries 3
apparent 'mismatched' case, i.e., hydrogenation of 18 in the and 4). Finally, the tert-butyl ester was cleaved selectively in
presence of (R,R)-Me-DuPHOS-Rh, it was also necessary to the presence of the acid-labile silyl group using silica in
increase the catalyst load (4 mol % added portionwise in com- refluxing toluene, thus affording the desired nucleosyl amino
parison to 1–2 mol % for the other reactions), and even under acid building blocks (S)-9 (94% yield) and (R)-9 (90% yield),
these modified conditions, full conversion could not be reached, each in diastereomerically pure form (Scheme 2). Overall, it can
resulting in a moderately reduced yield. For both transforma- be concluded that the omission of the 3-(N-BOM) protecting
tions of the isomeric mixture of 18, no hydrogenation of the group did not lead to improvements in the synthesis of 9, but
much less reactive E-isomer was observed, which is proven by rather made the sequence of Wittig–Horner olefination and
the high diastereoselectivities. If one takes into account that the asymmetric hydrogenation slightly less efficient. In contrast to
purity of the reactive Z-configured starting material Z-18 was uridine derivatives [48], thymidine analogues are significantly
only 91% (vide supra), it is possible to calculate yields of ca. more robust towards unwanted reduction of the 5,6-double bond
100% and 85% for products (S)-20 and (R)-20, respectively. in heterogeneously catalyzed hydrogenolysis reactions with
Overall, it can still be concluded though that 3-N-unprotected palladium catalysts. Therefore, the absence of the BOM group
didehydro amino acid 18 represented a less reactive substrate was not advantageous for the deprotection step following the
for the asymmetric hydrogenation key step, which was found to asymmetric hydrogenation reaction.
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Beilstein J. Org. Chem. 2015, 11, 50–60.
The second challenge on the way to target phosphoramidites 7 a mesylate, followed by treatment with sodium azide in DMF at
was the synthesis of protected 3'-amino-2',3'-dideoxyadenosine elevated temperature (110 °C). This protocol furnished 3'-azido
8 on a sufficient scale. Richert and Eisenhuth have published a derivative 25 in a moderate, but reliably obtained yield of 42%
comprehensive report on the synthesis of all four 3'-amino-2',3'- over 3 steps from 23. Azido nucleoside 25 was finally reduced
dideoxynucleosides with canonical bases [61]. We have decided to the 3'-amino analogue 8 by standard hydrogenation in 92%
to mainly follow their strategy for the synthesis of adenosine yield (Scheme 3).
derivative 8, though some modifications were applied
(Scheme 3). Starting from 2'-deoxyadenosine (22), N-6- With all building blocks 8, (S)-9 and (R)-9 in hand, the dimeric
benzoyl-5'-O-TBDMS-2'-deoxyadenosine (23) was prepared target structures could be constructed (Scheme 4). Using a stan-
[61] and subjected to oxidation of the 3'-hydroxy functionality dard procedure for peptide coupling, suitably protected nucleo-
to a keto group with Dess–Martin periodinane (DMP). This was syl amino acids (S)-9 and (R)-9 were activated and reacted with
followed by reduction with sodium borohydride via nucleo- amine 8 to give bis-O-silylated NAA-linked A–T dimers (S)-26
philic attack of the keto group from the sterically less hindered and (R)-26 in yields of 71% and 68%, respectively. For the
α-face, therefore resulting in the formation of the 3'-xylo deriva- subsequent desilylation reaction, several reaction conditions
tive 24. A tight control of the reaction conditions, i.e., amounts were tested, among them acidic silyl ether cleavage with
of reagents, reaction time and temperature, proved to be impor- hydrochloric acid in methanol or treatment with triethylamine
tant for this transformation (see Supporting Information File 1). trihydrofluoride (3HF•NEt3). However, the only successful
It was observed that the time period needed for the oxidation method was the conversion of both epimers of 26 with ammoni-
step was dependent on the scale of the reaction and that the um fluoride in methanol at elevated temperature, which
sensitive keto intermediate decomposed when this time period afforded diols (S)-27 and (R)-27, each in 66% yield. DMTr
was unreasonably exceeded. The obtained product 24 still protection under standard conditions then provided intermedi-
contained minor amounts of aromatic byproducts from the ates (S)-28 and (R)-28 in yields of 72% and 81%, respectively.
oxidizing agent, which were difficult to remove by column Finally, phosphitylation of the 3'-hydroxy group gave target
chromatography at this stage. Therefore, this material was phosphoramidites (S)-7 (57% yield) and (R)-7 (74% yield), each
employed in subsequent transformations without further with defined stereochemistry at the 6'-position. For this reac-
attempts to remove the aforementioned impurities. Several tion, 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite
methods are known to perform the nucleophilic displacement at 29 was used under slightly acidic conditions, i.e., in the pres-
the 3'-position of the 3'-xylo intermediate with azide as a nucle- ence of the activator 4,5-dicyanoimidazole (DCI, Scheme 4).
ophile [61-65]. We have found that robust results for this reac- The alternative method for the introduction of the phosphor-
tion could be achieved by activation of the 3'-hydroxy group as amidite functionality, i.e., treatment with the respective
Scheme 3: Synthesis of protected 3'-amino-2',3'-dideoxyadenosine 8.
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Beilstein J. Org. Chem. 2015, 11, 50–60.
Scheme 4: Synthesis of target phosphoramidites (S)-7 and (R)-7 and of two NAA-modified DNA oligonucleotides 30, 31 (DCI = 4,5-
dicyanoimidazole); sites of NAA-modifications in the oligonucleotides are indicated as x (6'S) and y (6'R), all other linkages were native phosphates.
chlorophosphite in the presence of a base, resulted in unwanted isolated and identified by ESI mass spectrometry. The sequence
concomitant cleavage of the Fmoc group.
of 30 and 31 resembled the sequence of model oligonucleotides
32 and 33 prepared for our initial studies on the NAA-modifica-
In order to demonstrate their principle synthetic versatility, tion [38] (Scheme 4). This strategy will enable systematic future
'dimeric' phosphoramidites (S)-7 and (R)-7 were employed for studies on the potential influence of the base sequence on the
the automated synthesis of two DNA oligonucleotides 30 and properties of NAA-modified oligonucleotides.
31, each bearing two NAA-modifications with defined stereo-
chemistry at the NAA-linkage ((6'S) or (6'R)) and therefore Conclusion
displaying partially zwitterionic backbone structures. After In summary, we have successfully accomplished the stereose-
assembly on the synthesizer and base-mediated cleavage from lective synthesis of 'dimeric' A–T phosphoramidite building
the solid support, the desired full-length products were purified, blocks (S)-7 and (R)-7 for the preparation of novel NAA-modi-
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Beilstein J. Org. Chem. 2015, 11, 50–60.
fied DNA oligonucleotides with partially zwitterionic back- doctoral fellowship of the Studienstiftung des deutschen
bone structures. The required nucleosyl amino acid intermedi- Volkes.
ates (S)-9 and (R)-9 were synthesized from thymidine in overall
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Supporting Information
Supporting Information features preparation, analytical data
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Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (DFG, grant
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