Synthesis of aldehyde-linked nucleotides and DNA and their bioconjugations with lysine and peptides through reductive amination

Article abstract of DOI:10.1002/chem.201103270

5-(5-Formylthienyl)-, 5-(4-formylphenyl)- and 5-(2-fluoro-5-formylphenyl) cytosine 2′-deoxyribonucleoside mono- (dC^RMP) and triphosphates (dC^RTP) were prepared by aqueous Suzuki-Miyaura cross-coupling of 5-iodocytosine nucleotides with the corresponding formylarylboronic acids. The dC^RTPs were excellent substrates for DNA polymerases and were incorporated into DNA by primer extension or PCR. Reductive aminations of the model dC^RMPs with lysine or lysine-containing tripeptide were studied and optimized. In aqueous phosphate buffer (pH 6.7) the yields of the reductive aminations with tripeptide III were up to 25 %. Bioconjugation of an aldehyde-containing DNA with a lysine-containing tripeptide was achieved through reductive amination in yields of up to 90 % in aqueous phosphate buffer. Joining forces: DNA-peptide conjugation through reductive amination of aldehyde-modified DNA with lysine-containing peptides was developed (see scheme). Formylaryl-linked dCTP derivatives were prepared by cross-coupling and incorporated into DNA by polymerases. The reductive amination of aldehyde-modified nucleotides and DNA was studied and optimized. Copyright

Full text of DOI:10.1002/chem.201103270

DOI: 10.1002/chem.201103270
Synthesis of Aldehyde-Linked Nucleotides and DNA and Their
Bioconjugations with Lysine and Peptides through Reductive Amination
Veronika Raindlovꢀ,[a] Radek Pohl,[a] and Michal Hocek*[a, b]
Abstract: 5-(5-Formylthienyl)-, 5-(4-
formylphenyl)- and 5-(2-fluoro-5-for-
were incorporated into DNA by primer
extension or PCR. Reductive amina-
tions of the model dCRMPs with lysine
or lysine-containing tripeptide were
studied and optimized. In aqueous
phosphate buffer (pH 6.7) the yields of
the reductive aminations with tripep-
tide III were up to 25%. Bioconju-
gation of an aldehyde-containing DNA
with a lysine-containing tripeptide was
achieved through reductive amination
in yields of up to 90% in aqueous
phosphate buffer.
mylphenyl)cytosine
2’-deoxyribonu-
cleoside mono- (dCRMP) and triphos-
phates (dCRTP) were prepared by
aqueous Suzuki–Miyaura cross-cou-
pling of 5-iodocytosine nucleotides
with the corresponding formylarylbor-
onic acids. The dCRTPs were excellent
substrates for DNA polymerases and
Keywords: aldehydes · DNA · nu-
cleotides · peptides · reductive ami-
nations
Introduction
An aldehyde function is a very attractive group because
of its high and specific reactivity with diverse reagents.
However, only several examples of nucleobase-linked alde-
hyde-modified ONs are known;[9–12] these are either pre-
pared directly or indirectly by click reaction with azide de-
rivatives of reducing sugars,[4f] or by introduction of 2,3-di-
hydroxypropyl or 3,4-dihydroxypyrrolidine moieties[13,14] fol-
lowed by oxidative cleavage of the vicinal diols to (di)alde-
hydes. Kittaka et al.[12] reported several examples of DNA–
protein cross-links through reductive amination of 5- or 6-
formyluracil. Sugar-linked aldehyde- or ketone-modified nu-
cleic acids have been more frequently explored;[1c,15] these
have often served for conjugation through Schiff base, hy-
drazone or oxime formation or reductive amination. Recent-
ly, we have reported a facile and efficient construction of
nucleobase-linked aldehyde-modified DNA in two steps
(cross-coupling of a nucleoside triphosphate with formylthi-
enylboronic acid followed by polymerase incorporation) and
its further conjugation or staining by hydrazone formation
in water.[16] The efficiency of this approach prompted us to
explore the potential of the (now) easily available formyl-
aryl–nucleobase linked DNA for conjugation with lysine
and lysine-containing peptides through reductive amination.
Bioconjugation of nucleic acids with other biomolecules or
unnatural compounds is of great importance in medicinal
chemistry and chemical biology (e.g., for delivery of oligo-
nucleotides)[1] or even in nanotechnology and material scien-
ces.[2] In most cases, the (bio)molecule is linked to a sugar or
phosphate at either the 3’ or 5’ end of an oligonucleotide
(ON).[3] Linkage through a nucleobase offers better pro-
grammability of the position(s) and number of modifica-
tions, and their exposition to the major groove in DNA. It is
usually done by attachment of a reactive group to a nucleo-
base in a nucleotide building block, incorporation to an ON
and post-synthesis attachment of the target molecule. Intro-
duction of an alkyne, alkene, azide or diene group or a cys-
teine residue either by chemical phosphoramidite synthesis
or by enzymatic polymerase synthesis has been achieved
and the modified DNA has been used for [2+3]-dipolar cy-
cloadditions (triazole or isoxazoline formation),[4,5] Stauding-
er ligation,[6] Diels–Alder reactions[7] or native peptide liga-
tions.[8]
[a] V. Raindlovꢀ, Dr. R. Pohl, Prof. Dr. M. Hocek
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Gilead Sciences & IOCB Research Center
Flemingovo nam. 2, 16610 Prague 6 (Czech Republic)
Fax : (+420)220-183-559
Results and Discussion
For the bioconjugation or cross-linking of DNA with natural
unmodified peptides or proteins, a reductive amination of
an aldehyde group with nucleophilic amino group of lysine
was envisaged. For the construction of the aldehyde-linked
DNA,[16] we planned to use the above-mentioned two-step
procedure[17] consisting of a cross-coupling of formylarylbor-
onic acids with halogenated 2’-deoxyribonucleoside triphos-
phates (dNTPs) followed by polymerase incorporation[18] by
E-mail: hocek@uochb.cas.cz
Homepage: http://www.uochb.cas.cz/hocekgroup
[b] Prof. Dr. M. Hocek
Department of Organic and Nuclear Chemistry
Faculty of Science, Charles University in Prague
Hlavova 8, 12843 Prague 2 (Czech Republic)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103270.
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FULL PAPER
primer extension (PEX) or polymerase chain reaction
(PCR). In this study, we focused on modifications of 2’-de-
oxycytidines bearing three different formylaryl groups at the
position 5 to study the electronic effects of the aryl group on
the reactivity of the aldehyde. At first, the synthesis and re-
ductive aminations were tested and optimized on the corre-
sponding nucleoside monophosphates (dNMPs) as models
for ONs and then the procedures were applied for the syn-
thesis of the modified dNTPs, their incorporations to DNA
and its conjugations with a model peptide.
were further used for the study of reductive aminations
(vide infra).
The corresponding aldehyde-modified nucleoside triphos-
phates (dCRTPs) were prepared (Scheme 1) analogously
from dCITP (2). The Suzuki cross-coupling reactions of 2
with boronic acids 3a–c gave the desired products dCFTTP
(5a),[16] dCFPhTP (5b) and dCFFTP (5c) in 65, 40 and 30%
yields, respectively, after isolation by semipreparative
HPLC, conversion to sodium salts by ion exchange and
freeze-drying.
The desired modified cytidine monophosphates (dCRMPs)
bearing reactive aldehyde groups were prepared by aqueous
Suzuki cross-coupling reaction of iodinated nucleoside
monophosphate dCIMP (1) with three different boronic
acids: 5-formylthiophene-2-boronic acid (3a), 4-formylphen-
yl boronic acid (3b) and 2-fluoro-5-formylphenyl boronic
acid (3c; Scheme 1). The reactions of dCIMP (1) with 3a–c
were performed in ACN/H2O in the presence of Cs2CO3,
These modified dCRTPs (5a–c) served as substrates for
polymerase incorporation into DNA. Seven different DNA
polymerases were tested in PEX or PCR experiments. The
enzymatic incorporation of dCRTPs (5a–c) in PEX was stud-
ied by using Pwo, KOD XL, DeepVent
(exoÀ), DeepVent,
ACHTUNGTRENNUNG
DyNAzyme II and Vent
AHCTUNGTRENNUNG
different templates (a 19-mer comprising single modification
and a 31-mer with four modifications in separate positions).
All of the tested dCRTPs were successfully and efficiently
incorporated into DNA by using both types of template, and
all the above-mentioned polymerases gave full-length alde-
hyde modified 19- or 31-mer ONs (Figure 1a and b; Fig-
ures S1–S3 in the Supporting Information). The thermal de-
naturation study (Table S5 in the Supporting Information)
showed slight destabilization of the duplex by formylthienyl
or formylphenyl (À0.25 or À0.758C per modification) and
significant (À2.758C per modification) destabilization for
the fluoroformylphenyl groups.
PdACHTUNGTRENNUNG(OAc)2 and tris(3-sulfonatophenyl)phosphine sodium salt
(TPPTS). The reaction with boronic acids 3a and b proceed-
ed smoothly at 100 or 908C, whereas the fluorinated boronic
acid 3c was less stable and the reaction temperature was de-
creased to 758C. The corresponding aldehyde-modified nu-
cleoside monophosphates dCFTMP (4a), dCFPhMP (4b) and
dCFFMP (4c) were isolated in good yields of 70–80% and
In order to compare the efficiency of incorporation of the
aldehyde-modified dCRTPs with the natural dCTP, we also
Figure 1. Primer extension and PCR incorporation of dCFTTP (5a),
dCFPhTP (5b) and dCFFTP (5c). a) PEX, 19-mer template, KOD XL
DNA polymerase. C-: dGTP; CFT: dCFTTP (5a), dGTP; CFPh: dCFPhTP
(5b), dGTP; CFF: dCFFTP (5c), dGTP. b) PEX, 31-mer template, DyNA-
zyme DNA polymerase; c) PCR, 297-mer template, KOD XL DNA poly-
merase. b) and c) C-: dTTP, dATP, dGTP; CFT: dCFTTP (5a), dTTP,
dATP, dGTP; CFPh: dCFPhTP (5b), dTTP, dATP, dGTP; CFF : dCFFTP
(5c), dTTP, dATP, dGTP. P: primer; L: oligonucleotide ladder; (+): natu-
ral dNTPs.
Scheme 1. Reagents and conditions: 1 or 2, 3a–c, Cs2CO3, PdACTHNURTGNENG(U OAc)2,
TPPTS, ACN/H2O. dCFTMP (4a): 1008C, 1 h; dCFTTP (5a): 1008C, 1 h;
dCFPhMP (4b): 908C, 2 h; dCFPhTP (5b): 908C, 30 min; dCFFTP (4c):
758C, 1.5 h; dCFFTP (5c): 758C, 30 min.
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M. Hocek et al.
performed a simple kinetic study in the single nucleotide
PEX experiment using two selected DNA polymerases
88%) and the side-products 11a–c were formed in negligible
amounts. On the other hand, in 10% glycerol or phosphate
buffer, the reduction of the aldehyde was the major reaction
(yields of 11a–c, 28–40%) and the desired products 6a–c
were only isolated in 18–33% yields. The yields of the reac-
tions of formylthienyl derivative 4a were significantly lower
than those of the two formylphenyl derivatives 4b,c.
Analogous reactions with lysine (II) were more complicat-
ed by the fact that both a- and e-amino groups can react in
the reductive amination with an aldehyde. Therefore, two
types of products 7a–c (e-) and 8a–c (a-) were observed in
all the reductive aminations of 4a–c. Moreover, in the reac-
tion of 4b with II in methanol, formation of a minor (3%)
compound 12b was observed as the product of double re-
ductive aminations at both amino groups (Scheme 3). The
combined yields of both reductive amination products (7+
8) were comparable or slightly lower in organic solvents
compared to acetylated lysine (I) but in aqueous solutions
the conversions of reductive aminations were slightly higher.
In methanol or glycerol, the ratio of e- to a-substituted
products 7a–c to 8a–c were 5:1 to 2:1, whereas in aqueous
glycerol or phosphate buffer the a-substituted products pre-
vailed (1:1 to 1:5).
The reactions of 4a–c with the acetylated tripeptide am-
ide III typically gave the desired products of the reductive
amination 9a–c (yields 22–52% in organic solvents and 10–
25% in aqueous solutions) accompanied by either hydroxy-
methylcytosine byproducts 11a–c (in water) or deacetylated
products of reductive amination 10a–c (in organic solvents).
In the reaction of 4a with III in glycerol, a minor byproduct
13a was isolated in 13% yield as a product of deacetylation
of III followed by reductive amination at the amino group
at the N terminus of the peptide (Scheme 3).
In general, all the reductive aminations gave mixtures of
products and the yields were moderate to good (but never
quantitative). Obviously, in methanol and glycerol, the
yields of the products of the reductive aminations were
higher than in aqueous glycerol or aqueous phosphate
buffer. However, only the aqueous solutions are applicable
for the intended bioconjugations of nucleic acids with pep-
tides. The formylphenyl derivative 4b gave the highest
yields of reductive aminations with peptide III in the phos-
phate buffer at pH 6.7, which are the conditions suitable for
reactions of nucleic acids and peptides. Therefore, we select-
ed the 5-(4-formylphenyl)cytosine triphosphate, dCFPhTP
(5b) for further studies of the conjugations.
(KOD XL and DeepVentACTHNUTRGNEUNG
(exoÀ)). The incorporation of for-
mylthienyl- and formylphenyl-linked dCFTTP (5a) and
dCFPhTP (5b), respectively, proceeded with a rate compara-
ble with unmodified dCTP and was finished within 30 sec-
onds. The incorporation of fluorinated dCFFTP (5c) took
2 min to completion. The enzymatic incorporation of
ACTHNUTRGNEUNG
dCRTPs by DeepVent(exoÀ) DNA polymerase proceeded
with a similar rate and efficiency, but with extension of the
reaction times (>5 min) formation of longer products by
non-templated incorporation of one or two more nucleo-
tides was observed (Figures S7–S9 in the Supporting Infor-
mation).
Then we tested the aldehyde-modified dCRTPs (5a–c) for
the amplification of DNA by PCR with three templates (98,
297 and even 1162 nucleotides [nt]) using selected DNA
polymerases (Phusion, VentACHTNUGRTNEUNG
(exoÀ) and KOD XL). All the
PCR amplifications with dCFTTP (5a) and dCFPhTP (5b)
were successful with all three polymerases and templates
(Figure 1c and Figures S4–S6 in the Supporting Informa-
tion) and clean (only slightly smeared) spots of the full-
length products were observed on the gels in all cases.
dCFFTP (5c), however, was a far worse substrate in PCR re-
actions. The spots of the 98 and 287 nt DNA products were
rather smeared and the 1162-mer was not detected at all.
This is consistent with the lower efficiency of dCFFTP (5c)
revealed in the kinetic studies and thermal denaturation
study (vide supra).
The next goal was to develop the procedure for the conju-
gation of the aldehyde-modified DNA through reductive
amination. This reaction was first tested and optimized on
all the three model dCRMPs (4a–c) by using two amino
acids, acetylated and free lysine (Ac-Lys-NH2·HCl, I, and
Lys·HCl, II) and one synthetically prepared lysine-contain-
ing tripeptide (Ac-Ala-Lys-Ala-NH2, III). Reductive amina-
tion in water is challenging due to unfavorable equilibrium
in the Schiff-base formation as it has been repeatedly shown
in dynamic combinatorial chemistry studies.[19] Therefore,
we tested the reactions in methanol, glycerol, aqueous glyc-
erol (10%) and phosphate buffer (Scheme 2, Table 1). Sev-
ACTHNUTRGNEUNG
eral reducing agents (Na2S2O4,[20] NaBH(OAc)3,[21] H2/Pd-
[23]
C,[22] NaBH4/H3BO3 and NaBH3CN[11]) were tested under
different conditions but, in most cases, the conversions were
very low and only the reduction of the aldehydes to hy-
droxymethylcytosines 11a–c was observed. Only the use of
NaBH3CN gave acceptable conversions of the reductive
aminations, and therefore, this reagent was used in the sys-
tematic screening of the conditions. As expected, both the
conversions and the selectivities for reductive aminations
versus aldehyde reductions were strongly solvent-dependent
(Table 1 and Tables S1–S3 and S4 in the Supporting Infor-
mation).
To test the conjugations of aldehyde-modified nucleic
acids with the peptide III, we incorporated one or four
modified nucleotides using dCFPhTP (5b) with either the 19-
or 31-mer template in the presence of DeepVentACHTUNGTRENNUNG
(exoÀ)
DNA polymerase under standard the conditions discussed
above. Then the resulting modified DNA, either unpurified
or after purification, was treated with the peptide III in the
presence of NaBH3CN in phosphate buffer. The reaction
with III (3 equiv) showed only very minor conjugate forma-
tion. Only further increase in the excess of III (to 50-fold)
gave high conversion of the conjugation. Figure 2 shows the
The reactions of 4a–c with acetylated lysine (I) usually
gave mixtures of the desired conjugates 6a–c and the re-
duced hydroxymethyl derivatives 11a–c. In methanol or
glycerol, the yields of 6a–c were good to excellent (55–
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Aldehyde-Linked Nucleotides and DNA
FULL PAPER
Scheme 2. Reductive aminations of 4a–c with amino acids I, II and peptide III (3 equiv) in the presence of NaBH3CN (1 equiv).
Table 1. Yields of the reductive aminations of 4a–c with amino acids I, II and peptide III.
Ac-Lys-NH2·HCl (I)
Lys·HCl (II)
Yield [%]
Ac-Ala-Lys-Ala-NH2 (III)
Solvent
Product
Yield [%]
Product
(7x:8x)[a]
Product
Yield [%]
À
Ar CH=O
methanol
55[b]
63[b]
19[b]
18[b]
54[c]
51[c]
25[c]
26[b]
4:1
2:1
1:2
1:1
29[c,e]
36[c]
10[c]
13[b]
99% glycerol
10% glycerol
6a
6b
6c
7a+8a
9a
9b
9c
2À
PO4 buffer[d]
methanol
71[b]
88[b]
28[b]
24[b]
44[c]
50[c]
53[c]
26[b]
5:1
2:1
1:1
1:2
52[c,f]
51[c,g]
23[c]
99% glycerol
10% glycerol
7b+8b
7c+8c
2À
PO4 buffer[d]
25[b]
methanol
76[b]
79[b]
33[b]
25[b]
77[c]
74[c]
35[c]
44[b]
5:1
2:1
1:1
1:5
24[c,h]
22[c]
23[c]
22[b]
99% glycerol
10% glycerol
2À
PO4 buffer[d]
[a] x=a, b or c. [b] 48 h. [c] 24 h. [d] phosphate buffer (50 mm, pH 6.7). [e] 10a (6%). [f] 10b (14%). [g] 10b (6%). [h] 10c (5%).
results of the reductive aminations of the 19-mer PEX prod-
uct containing one formylphenyl modification with different
modes of purification of the PEX products and/or the reduc-
tive amination products. In all cases, the spot of the PEX
product (lane CFPh) almost fully disappeared and an intense
new band of the DNA–peptide conjugate was observed with
slower mobility (lanes 1–4). The conversions of the reduc-
tive aminations were calculated from the PAGE by using
the ImageJ quantification program (Figure S10 in the Sup-
porting Information). The results showed that with unpuri-
fied PEX product the conversion was about 80% whereas
with purified PEX product, the yield was 90%. Final purifi-
cation of the DNA–peptide conjugate (NucAway spin col-
umns) did not further improve the purity. The formation of
this DNA–peptide conjugate was confirmed by MALDI-
ToF MS spectrometry (found m/z 6369.5, calcd 6368.2; Fig-
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M. Hocek et al.
Conclusion
An efficient and straightforward synthesis of three different
5-formylaryl-modified cytidine mono- and triphosphates was
developed by using the aqueous cross-coupling reactions of
5-iodocytosine nucleotides with formylarylboronic acids.
The dCRTPs were excellent substrates for DNA polymerases
and were incorporated into DNA by means of PEX, and
two of them also by PCR, with an efficiency close to natural
dCTP. In this way, different aldehyde-modified DNAs were
prepared, ranging from short ONs bearing one or several
modifications up to large DNA duplexes (100–1200 nt) bear-
ing many aldehyde groups (40–580) at specific positions.
The reactivity toward reductive aminations with lysine, N-
protected lysine and a lysine-containing tripeptide were first
tested on model nucleotides dCRMPs (4a–c) and the effect
of solvent was studied. Obviously, the reductive aminations
proceeded much better in methanol or glycerol than in
aqueous mixtures. In the case of lysine, the solvent also in-
fluenced the ratio of e- to a-amino group chemoselectivity.
In phosphate buffer, as the only suitable medium for bio-
conjugations, the maximum yields of reductive aminations
with tripeptide III were about 25% for formylphenyl deriva-
tive 4b. This modification was then incorporated into DNA
and the PEX product was subjected for the reductive amina-
tion with tripeptide III. This reaction gave 80–90% conver-
sion but only in the presence of large (50-fold) excess of the
tripeptide. Apparently, the efficiency of the reductive amina-
tion cannot match the success of Cu-mediated triazole for-
mation used in click chemistry.[4] However, the reductive
amination can be performed with native peptides and pro-
teins containing natural lysine (no incorporation of any un-
natural amino acid is needed in contrast to the click reac-
tion).[4] Therefore, we believe that this method has promis-
ing potential in nucleic acid–peptide or –protein conjuga-
tions. In particular, cross-linking[24] with some sequence-spe-
cific DNA-binding proteins could be improved by specific
interactions between the DNA and protein. On the other
hand, the high natural abundance of lysine in proteins could
impair the specificity of the bioconjugation. Studies of the
reductive aminations of aldehyde-modified ONs with longer
peptides and proteins, as well as their applications in cross-
linking assays for protein binding are under way in our labo-
ratory.
Scheme 3. Minor byproducts in reductive amination reactions.
Figure 2. a) Scheme of peptide–DNA conjugation via
linker. b) PAGE analysis of PEX and the DNA–peptide conjugation by
using the 19-mer template, DeepVent
(exoÀ) DNA polymerase and pep-
tide III; P: primer; +: natural dNTPs; CFPh dCFPhTP (5b), dGTP;
a phenyl unit
AHCTUNGTRENNUNG
:
lanes 1–4: dCFPhTP (5b), dGTP, Ac-Ala-Lys-Ala-NH2 (50 equiv),
NaBH3CN; lane 1: no purification; lanes 2, 3: PEX product purification
(QIAquick nucleotide removal kit); lanes 2, 4: DNA–peptide conjugate
purification (NucAway Spin columns).
Experimental Section
General chemistry: NMR spectra were measured on
a Bruker
Avance 500 at 500 MHz for 1H and 125.7 MHz for 13C, or on Bruker
Avance at 600 MHz for 1H and 150.9 MHz for 13C, instruments in D2O
(reference to dioxane as internal standard, dH =3.75 ppm, dC =
67.19 ppm) or in [D4]methanol (reference to TMS as internal standard).
Chemical shifts are given in ppm (d scale), coupling constants (J) in Hz.
Complete assignment of all NMR signals were achieved by using a com-
bination of H,H COSY, H,C HSQC and H,C HMBC experiments. Semi-
preparative separation of nucleoside triphosphates and monophosphates
was performed by HPLC on a column packed with 10 mm C18 reversed
ure S12 in the Supporting Information). In the case of the
31-mer PEX product containing four aldehydes, the reduc-
tive amination was quantitative (Figure S11 in the Support-
ing Information) but a mixture of several products (presum-
ably of single and multiple conjugation) was observed.
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Aldehyde-Linked Nucleotides and DNA
FULL PAPER
phase (Phenomenex, Luna C18 (2)). IR spectra were measured either on
Bruker Alpha FT-IR spectrometer by using ATR technique or by using
KBr tablets. Mass spectra were measured by ESI. High resolution mass
spectra were measured on a LTQ Orbitrap XL (Hermo Fischer Scientif-
ic) spectrometer by using ESI ionization technique. Mass spectra of func-
tionalized DNA were measured by MALDI-ToF, Reflex IV (Bruker)
Compound 9b: This was prepared from dCFPhMP (4b) according to the
general procedure (method C) in phosphate buffer (50 mm, pH 6.7).
After being stirred for 48 h the product was isolated as white powder
(7 mg, 25%): 1H NMR (499.8 MHz, D2O, refdioxane =3.75 ppm): d=1.35
(d, 3H, J3,2 =7.2 Hz, H-3-AcAla), 1.39 (d, 3H, J3,2 =7.2 Hz, H-3-AlaNH2),
1.46 (m, 2H, H-4-Lys), 1.73 (m, 2H, H-5-Lys), 1.76, 1.85 (2ꢂbm, 2ꢂ1H,
with nitrogen laser. UV/Vis spectra were measured on
a
Varian
H-3-Lys), 2.00 (s, 3H, CH3CO), 2.33 (ddd, 1H, Jgem =14.1 Hz, J2’b,1’
7.9 Hz, J2’b,3’ =6.2 Hz, H-2’b), 2.43 (ddd, 1H, Jgem =14.1 Hz, J2’a,1’ =6.1 Hz,
2’a,3’ =3.4 Hz, H-2’a), 3.07 (t, 2H, J6,5 =7.8 Hz, H-6-Lys), 3.85, 3.88 (2ꢂdt,
2ꢂ1H, gem =11.3 Hz, H,P =J5’,4’ =5.3 Hz, H-5’), 4.15 (td, 1H, J4’,5’
5.3 Hz, J4’,3’ =3.4 Hz, H-4’), 4.21 (s, 2H, CH2N), 4.24 (q, 1H, J2,3 =7.2 Hz,
H-2-AcAla), 4.27 (q, 1H, J2,3 =7.2 Hz, H-2-AlaNH2), 4.31 (dd, 1H, J2,3
8.7, 5.7 Hz, H-2-Lys), 4.51 (dt, 1H, J3’,2’ =6.2, 3.4 Hz, J3’,4’ =3.4 Hz, H-3’),
6.34 (dd, 1H, J1’,2’ =7.9, 6.1 Hz, H-1’), 7.51 (m, 2H, H-o-phenylene), 7.56
(m, 2H, H-m-phenylene), 7.76 ppm (s, 1H, H-6); 13C NMR (125.7 MHz,
D2O, refdioxane =69.3 ppm): d=19.16 (CH3-3-AcAla), 19.38 (CH3-3-
AlaNH2), 24.26 (CH3CO), 24.89 (CH2-4-Lys), 28.03 (CH2-5-Lys), 33.01
(CH2-3-Lys), 41.66 (CH2-2’), 49.89 (CH2-6-Lys), 52.15 (CH-2-AlaNH2),
=
CARY 100 Bio spectrophotometer at room temperature. Known starting
compounds were either purchased from suppliers and used without any
further treatment (5-formylthiophene-2-boronic acid (3a), 4-formylphen-
yl boronic acid (3b) and 2-fluoro-5-formylphenyl boronic acid (3c) from
Frontier Scientific) or prepared by literature procedures (compounds
dCIMP (1),[16] dCITP (2),[17c] dCFTMP (4a)[16] and dCFTTP (5a)[16]).
Preparation of dCFPhTP (5b) by the Suzuki cross-coupling reaction: A
water/acetonitrile mixture (2:1, 1 mL) was added through a septum to an
argon-purged vial containing 5-iodo-2’-deoxycytidine triphosphate
(dCITP, (2); 0.047 g, 0.071 mmol), 4-formylphenyl boronic acid (3b;
3 equiv, 0.017 g, 0.213 mmol) and Cs2CO3 (5 equiv, 0.062 g, 0.355 mmol).
J
J
J
=
=
52.52 (CH-2-AcAla), 53.59 (CH2NH), 55.93 (CH-2-Lys), 66.35 (d, JC,P
=
After the solids had dissolved, a solution of PdACTHNURTGNENG(U OAc)2 (0.9 mg, 10 mol%)
4.4 Hz, CH2-5’), 73.94 (CH-3’), 88.69 (CH-1’), 88.80 (d, JC,P =8.5 Hz, CH-
4’), 112.74 (C-5), 132.83 (CH-o-phenylene), 133.18 (CH-m-phenylene),
134.96 (C-p-phenylene), 136.00 (C-i-phenylene), 142.71 (CH-6), 159.81
(C-2), 167.17 (C-4), 176.30 (C-1-Lys), 176.92 (COCH3), 178.29 (C-1-
AcAla), 180.31 ppm (C-1-AlaNH2); 31P{1H} NMR (202.3 MHz, D2O): d=
4.50 ppm; IR (KBr): v˜ =3387, 2937, 1652, 1543, 1479, 1416, 1374, 1313,
1265, 1073, 975 cmÀ1; MS (ESI+): m/z (%): 725.3 (12) [M+H]+, 747.3
(63) [M+Na]+, 769.3 (52) [MÀ1H+2Na]+, 770.2 (19) [M+2Na]+, 791.2
(10) [MÀ2H+3Na]+; HRMS (ESI+): m/z [M+H]+ calcd for
C30H46O11N8P: 725.30182, found 725.30218; UV/Vis (H2O, 100 mm) lmax1
and TPPTS (5 equiv, 0.011 g, 0.020 mmol) in water/acetonitrile mixture
(2:1, 1 mL) was injected into the reaction mixture and the resulting
sample was stirred at 908C for 30 min. The reaction mixture was extract-
ed with CHCl3 (2ꢂ4 mL). The water layer containing the product was
concentrated by evaporation and product was isolated by semiprepara-
tive HPLC on a C18 column with the use of a linear gradient starting
from TEAB (0.1m; triethylammonium bicarbonate) in H2O/MeOH 3:1
to 0.1m TEAB in H2O/MeOH 1:1 as eluent. Several co-distillations with
water, conversion to sodium salt form (Dowex 50WX8 in Na+ cycle) fol-
lowed by freeze drying from water, gave the product dCFPhTP (5b) as a
white powder (0.009 g, 40%). 1H NMR (499.8 MHz, D2O, refdioxane
3.75 ppm, pD=7.1): d=2.37 (ddd, 1H, gem =14.1 Hz, 2’b,1’ =7.1 Hz,
2’b,3’ =6.4 Hz, H-2’b), 2.46 (ddd, 1H, Jgem =14.1 Hz, J2’a,1’ =6.3 Hz, J2’a,3’
3.5 Hz, H-2’a), 4.12–4.20 (m, 2H, H-5’), 4.22 (m, 1H, H-4’), 4.63 (dt, 1H,
3’,2’ =6.4, 3.5 Hz, J3’,4’ =3.5 Hz, H-3’), 6.35 (dd, 1H, J1’2’ =7.1, 6.3 Hz, H-
=
(e)=238
(10126),
lmax2
(e)=277
(6193),
l
(e)=254 nm
(8459 molÀ1 dm3 cmÀ1).
J
J
J
=
Primer extension experiment (single incorporation): The reaction mix-
ture (20 mL) contained KOD XL DNA polymerase (Merc4biosciences,
Novagen, 2.5 UmLÀ1, 0.02 mL), dGTP (4 mm, 0.05 mL) and dCTP (4 mm,
0.05 mL), dCFTTP (5a), dCFPhTP (5b) or dCFFTP (5c; 4 mm, 1 mL), primer
(3 mm, 1 mL, Prim248 short: 3’-GGGTACGG CGGGTAC-5’) and 19-mer
template (3 mm, 1.5 mL, Oligo1C: 5’-CCCGCCCAT GCCGCCCATG-3’)
in KOD XL reaction buffer (2 mL) supplied by the manufacturer.
Prim248 short was labeled by the use of [g32P]-ATP according to standard
techniques. Reaction mixtures were incubated for 12 min at 608C in a
thermal cycler and were stopped by addition of stop solution (40 mL,
80% [v/v] formamide, 20 mm EDTA, 0.025% [w/v] bromophenol blue,
0.025% [w/v] xylene cyanol) and heated for 5 min at 958C. Reaction mix-
tures were separated by the use of a 12.5% denaturing PAGE. Visualiza-
tion was performed by phosphorimaging.
J
1’), 7.68 (m, 2H, H-o-phenylene), 7.89 (s, 1H, H-6), 8.05 (m, 2H, H-m-
phenylene), 9.98 ppm (s, 1H, CHO); 13C NMR (125.7 MHz, D2O,
refdioxane =69.3 ppm, pD=7.1): d=41.90 (CH2-2’), 67.92 (d, JC,P =5.7 Hz,
CH2-5’), 73.23 (CH-3’), 88.6 (d,
JC,P =8.8 Hz, CH-4’), 88.90 (CH-1’),
112.41 (C-5), 132.76 (CH-o-phenylene), 133.66 (CH-m-phenylene), 138.20
(C-p-phenylene), 141.97 (C-i-phenylene), 143.21 (CH-6), 159.72 (C-2),
166.85 (C-4), 198.77 ppm (CHO); 31P{1H} NMR (202.3 MHz, D2O,
refphosphatebuffer =2.35 ppm, pD=7.1): d=À21.70 (t, J=19.6 Hz, Pb), À10.99
(d, J=19.6 Hz, Pa), À6.57 ppm (bd, J=19.6 Hz, Pg); IR (KBr): v˜ =3428,
2435, 2366, 1742, 1700, 1652, 1603, 1490, 1367, 1312, 1159, 1085, 981 cmÀ1
;
MS (ESIÀ): m/z (%): 613.9 (20) [MÀNa]À, 658.2 (5) [MÀH+Na]À, 284.6
(66) [M+HÀ3Na]2À
,
512.0 (32) [M+2HÀNaÀHPO3Na]À, 490.1 (31)
Polymerase chain reaction (287nt): The PCR reaction mixture (20 mL)
contained KOD XL DNA polymerase (Merc4biosciences, Novagen,
2.5 UmLÀ1, 0.8 mL), DMSO (100%, 1 mL), formamide (5%, 1 mL), be-
taine (0.75m, 1 mL), TMAC (50 mm, 1 mL), natural dNTPs (10 mm,
0.5 mL) functionalized dCFTTP (5a), dCFPhTP (5b) or dCFFTP (5c)
(10 mm, 2 mL), primers (20 mm, 2 mL, PrimS1-HIV1: 5ꢃ-GATCACTCTT
TGGCAGCGAC CCCTCGTCAC-3ꢃ, and 20 mm, 2 mL, Prim S2-HIV1:
5ꢃ-TTAAAGTGCA GCCAATCTGA GTCAACAGAT-3ꢄ), and a 297-
mer template (74.02 ngmLÀ1, 0.3 mL, wt-HIV-1 PR: 5ꢃ-CCTCAGATCA
CTCTTTGGCA GCGACCCCTC GTCACAATAA AGATAGGGGG
GCAATTAAAG GAAGCTCTAT TAGATACAGG AGCAGATGAT
ACAGTATTAG AAGAAATGAA TTTGCCAGGA AGATGGAAAC
CAAAAATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG
ACAGTATGAT CAGATACTCA TAGAAATCTG CGGACATAAA
GCTATAGGTA CAGTATTAGT AGGACCTACA CCTGTCAACA
TAATTGGAAG AAATCTGTTG ACTCAGATTG GCTGCACTTT
AAATTTT-3ꢃ) in KOD XL reactions buffer (2 mL) supplied by the man-
ufacturer. Forty PCR cycles were run under the following conditions: de-
naturation for 1 min at 948C, annealing for 1 min at 608C, extension for
1 min at 728C, followed by a final extension step of 5 min at 728C. PCR
products were analyzed on a 1.3% agarose gel stained with GelRed (Bio-
tium, 10000ꢂ in H2O) in 0.5ꢂTBE buffer.
[M+3HÀ2NaÀHPO3Na]À; HRMS (ESIÀ): m/z [MÀNa]À calcd for
C16H17O14N3Na2P3: 613.97242, found 613.97192; UV/Vis (H2O, 100 mm)
lmax1 (e)=286 (3198), l (e)=254 nm (2146 molÀ1 dm3 cmÀ1).
General procedure for reductive aminations of dCRMPs (4a–c) with
amino acids I, II and peptide III (methods A, B, C): Amino acid or pep-
tide (3 equiv) was added in one portion to a stirred solution of aldehyde
derivative dCRMP (4a–c) in 2.5 mL of different solvents (MeOH, 99%
glycerol, 10% glycerol or 50 mm phosphate buffer, pH 6.7) under an at-
mosphere of argon. After 1 h, NaBH3CN (1 equiv) was added through a
syringe in the corresponding solvent (0.5 mL). After being stirred for
24 h (48 h) at room temperature under an atmosphere of argon the crude
products and also side-products were isolated by semipreparative HPLC
on a C18 column with the use of linear gradient of TEAB (triethylammo-
nium bicarbonate, 0.1m) in H2O to 0.1m TEAB in H2O/MeOH (1:1) as
eluent. Several co-distillations with water, conversion to sodium salt form
(Dowex 50WX8 in Na+ cycle) followed by freeze drying from water gave
the products and side-products as white powders. Method A: amino
acid I, 48 h, products 6a–c, byproducts 11a–c; method B: amino acid II,
24 h (48 h in phosphate buffer), products 7a–c, 8a–c, byproducts 11a–c,
12b; method C: peptide III, 24 h (48 h in phosphate buffer), products
9a–c, byproducts 10a–c, 11a–c, 13a.
Chem. Eur. J. 2012, 18, 4080 – 4087
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4085

M. Hocek et al.
DNA–peptide conjugation experiment: Six reaction mixtures (+, CFPh, 1,
2, 3, 4, 20 mL each) were prepared according to standard PEX procedure.
Ed. 2008, 47, 3442–3444; d) F. Seela, V. R. Sirivolu, P. Chittepu, Bio-
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2006, 128, 1398–1399.
TM(exoÀ) DNA polymerase
All reaction mixtures contained DeepVentR ACHTUNGRTENNUGN
(New England BioLabs, 2 UmLÀ, 4.5 mL), dGTP (4 mm, 0.15 mL) and
dCTP (4 mm, 0.15 mL), dCFPhTP (5b; 4 mm, 3 mL), primer (3 mm, 3 mL,
Prim248 short: 3’-GGGTACG GCGGGTAC-5’), and 19-mer template
(3 mm, 4.5 mL, Oligo1C: 5’-CCCGCCCATG CCGCCCATG-3ꢃ) in
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6883.
DeepVentACHTUNGTRENNUNG
(exoÀ) reaction buffer (2 mL) supplied by the manufacturer.
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Prim248 short was labeled by the use of [g32P]-ATP according to standard
techniques. Reaction mixtures were incubated for 30 min at 608C in a
thermal cycler and were stopped by addition of stop solution (12 mL,
80% [v/v] formamide, 20 mm EDTA, 0.025% [w/v] bromophenol blue,
0.025% [w/v] xylene cyanol) and heated 5 min at 958C. Samples 3 and 4
were purified by using the QIAquick nucleotide removal kit according to
standard techniques and eluted with phosphate buffer (50 mL, 50 mm,
pH 6.7). Then Ac-Ala-Lys-Ala-NH2, III (0.7m solution in 50 mm phos-
phate buffer, pH 6.7, 10.5 mL) and NaBH3CN (1.59m solution in 50 mm
phosphate buffer, pH 6.7, 1.5 mL) were added into each reaction mixture
(1–4). Such prepared mixtures were stirred in the thermal cycler at 258C
for 24 h/750 rpm. After this, reaction mixtures 2 and 4 were purified by
NucAwayTM spin columns. In addition, reaction mixture 1 was not puri-
fied. Finally, all reaction mixtures (+, CFPh, 1, 2, 3, 4) were separated by
the use of a 12.5% denaturing PAGE and visualized by phosphorimag-
ing.
Polyacrylamide gel electrophoresis: The PEX products containing load-
ing buffer (12 mL, 80% [v/v] formamide, 20 mm EDTA, 0.025% [w/v]
bromophenol blue, 0.025% [w/v] xylene cyanol) were subjected to verti-
cal electrophoresis in 12.5% denaturing polyacrylamide gel containing
1ꢂTBE buffer (pH 8) and urea (7m) at 45 mA for about 50 min. Gels
were dried (858C, 70 min) and the autoradiographs were visualized by
using a phosphorimager (Typhoon 9410, Amersham Biosciences).
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This work is a part of the research projects supported by the Academy of
Sciences of the Czech Republic (Z40550506). It was specifically support-
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www.chemeurj.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 4080 – 4087

Aldehyde-Linked Nucleotides and DNA
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Received: October 17, 2011
Published online: February 15, 2012
Chem. Eur. J. 2012, 18, 4080 – 4087
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