Synthesis and structural characterization of monomeric and dimeric peptide nucleic acids prepared by using microwave-promoted multicomponent reactions

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Synthesis and structural characterization of monomeric and

dimeric Peptide Nucleic Acid prepared by microwave-promoted

multicomponent reaction†

Reuben Ovadia^a, Aurélien Lebrun^b, Ivan Barvik^d, Jean-Jacques Vasseur^c, Carine Baraguey^cand

Karine Alvarez^a

Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

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A solution phase synthesis of Peptide Nucleic Acid monomers and dimers was developed by using microwave-promoted

Ugi multicomponent reactions. The mixture of a functionalized amine, a carboxymethyl nucleobase, paraformaldehyde

and an isocyanide as building blocks generates PNA monomers which are then partially deprotected and used in a second

Ugi 4CC reaction, leading to PNA dimers. Conformational rotamers were identified by NMR and MD simulations.

natural oligonucleotides as they show a high binding affinity to

complementary DNA and RNA sequences and display stability

towards enzymes which degrade peptides or nucleic acids. Many

practical applications of PNAs and PNA analogues as gene therapy

Introduction

In our search for efficient inhibitors of viral polymerases to control

infections originating from human viruses, we have recently

described as HCV polymerase inhibitors several ^5’-GC-^3’

phosphoramidate dinucleosides^{1, 2}. These active compounds were

rationally designed to hence their inhibition properties by blocking

the active site of the HCV polymerase.

drugs or molecular probes for diagnostics have attracted much

interest for over 20 years^{4, 5}

.

PNA oligomers are in most cases synthesized by solid phase peptide

assembly from N-protected monomeric building blocks, via

automated synthesis⁶. For short PNA sequences, liquid phase

synthesis is preferred and a variety of N-protected unit syntheses^{7, 8}

has been described with different strategies of protection for the

amino function of the N-terminus and for the amino function of the

nucleobases. Dömling and colleagues have described⁹, for the first

time in 1998, the original concept of combinatorial PNA synthesis¹⁰

to obtain PNA monomers and multimers by consecutive Ugi-4CC

We took advantage of this original « dimeric » approach to conceive

new compound family, (1) with higher binding to the active site of

the polymerase and to the RNA template, (2) with higher stability in

biological media and (3) easier to synthesize to afford molecular

diversity to target a panel of viral polymerases.

reactions. Several reports demonstrated the usefulness of Ugi-4CC

Peptide Nucleic Acids (PNAs) appeared as good candidates to reach

our goal. PNAs, described first in 1991 by Nielsen and his

colleagues³, are structural homologs of DNA and RNA with a

pseudo-peptide backbone of 2-amino-ethylglycine units to which

purine and pyrimidine nucleobases are linked via a methylene

carbonyl linkage. Oligomeric PNAs afford several advantages over

12

in the synthesis of PNA monomers^11,

in one-pot reaction, by

mixing a functionalized amine, a carboxymethyl nucleobase, an oxo-

component and an isocyanide.

Despite the originality of this strategy to generate PNA multimers

with a highly diverse substitution pattern, to date, only one

publication¹³describes the synthesis of protected TT/CT sequences

PNA dimers in solution.

We describe here an attractive synthetic strategy of PNA monomers

(T, C, A, G, U) prepared by one-pot Ugi-4CC reaction and dimers (TT,

AC, GC, GU, AG) prepared by two consecutive Ugi-4CC reactions

(Scheme 1). In order to obtain target compounds with high yields

and shorter reaction times, we turned our attention to microwave

irradiation (MWI). Complete NMR assignment of PNA monomers

and dimers was performed and allowed us to characterize

conformational rotamers.

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Scheme 1 Synthetic scheme of PNA dimers via two consecutive Ugi-4CC reactions

Results and discussion

Largely used in combinatorial chemistry, the Ugi-4CC plays a

significant role on the development of atom-economic and

synthetically effective syntheses. Ugi-4CC is also known to be one of

the most versatile tools for access to peptide like derivatives^14-16by

one-pot condensation of a functionalized amine, a carboxymethyl

nucleobase, an oxo-component and an isocyanide.

To conceive PNA monomers and dimers, we first designed the

different building blocks and the strategy of protection of the N-

protected units (Scheme 1 and Figure 1) to modulate N-terminus

and C-terminus patterns and offer different conditions of

deprotection. Our choices were also driven by the ease of access to

the designed building blocks. For the N-terminus, we chose

hydroxylamine protected by a tert-butyl-diphenylsilyl group 1b to

Fig. 1 Structure of the building blocks : 1 – functionalized amine, 2-

carboxymethyl nucleobase, 3- isocyanide

generate hydroxyl end or ethylene diamine protected by the acid

labile Boc group 1a and Mmt group 1d or by the Fmoc group 1c to

generate amino end. The carboxymethyl nucleobases 2a, 2c and 2d

were protected by base labile acyl groups. We designed also two

isocyanides able to perform oligomerization 3a and 3c and one

unable 3b.

The carboxymethyl nucleobases 2a, 2b, 2c and 2e were prepared in

three steps according to literature procedures^{17, 18}with 22%, 28%

and 31% global yield, respectively. Hydroxylamine 1b and ethylene

diamine 1a were synthesized according to the literature

20

procedures^19,

with 76% and quantitative yield, respectively.

Isocyanides 3a and 3c were prepared in 2 steps. Compound 1a and

1c were dissolved in ethyl formate and heated at reflux for 18 h to

furnish the corresponding formamides. Dehydration of formamides

with POCl₃in THF afforded isocyanides 3a and 3c with 80% and 38%

global yield, respectively.

Monomer synthesis

Commonly, the preparation in liquid phase of N-protected

monomers requires two steps: (1) the synthesis of the backbone

unit via reductive amination of a glycine ester with an N-protected

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aminoacetaldehyde, or via an alkylation for mono-protected Ugi-4CC is generally performed at high concentration (0.5 M to 2 M)

ethylenediamine with a bromoacetic acid ester, and (2) the linkage in protic solvent as methanol, ethanol or isopropanol at room

of the protected nucleobases via an amide bond using a coupling temperature, during 24 to 48 h. Using classical conditions, with 0.5

agent. For oligomer synthesis, the major drawback of this method is M concentration of starting material in equimolar mixture at rt

that prior preparation of each PNA monomer is needed. Likewise, during 24 h in MeOH, the compound 4a was obtained with 26%

any modification of the backbone requires the multi-steps yield and the byproduct 4a’ formation was around 6% (entry 1,

monomer synthesis and a complete resynthesis of the oligomer.

Table 1). The monomer precipitates in the medium when it is

formed to facilitate its recovery as a white powder by simple

With consecutive Ugi-4CC reaction method, the monomer is built filtration and washing. At higher concentration of starting material,

up along the elongation of the chain and the oligomer formation is until 2M, the yield was not improved due to the unfavorable

performed by cycle of coupling of nucleobase unit and then precipitation of some building blocks. The addition of 0.05 or 0.1

deprotection phase. This strategy can be used advantageously to equivalent of compound 1b and paraformaldehyde versus

introduce a diversity of building blocks during each cycle of compounds 2a and 3a, did not either improve the yield.

elongation.

By substituting the MeOH by isopropanol, the yield decreased

To probe the strategy to synthesize PNA monomers by Ugi-4CC (entry 2, Table 1) and was not widely improved neither even if the

previously proposed by P. Xu¹³, the Ugi product 4a (guanine PNA reaction was continued longer (48h) (entry 3, Table 1).

monomer model) served our exploration to study the effect of The effect of the reaction temperature was then evaluated. At 60

concentration of building blocks, solvent, reaction time, °C, the compound 4a was formed faster and reaction time was

temperature, MW irradiation, and pre-incubation of protected reduced by half. However, these conditions were in favor of the

hydroxylamine 1b and paraformaldehyde. We mixed protected formation of the byproduct 4a’ (36% yield) and unfavorable for the

hydroxylamine

1b,

carboxymethyl

nucleobase

2a, formation of the expected compound 4a (23% yield) (entry 4, Table

paraformaldehyde and isocyanide 3a and modulated experimental 1).

conditions. Conditions and yields are gathered in Table 1. The aim In order to reduce the formation of the byproduct 4a’, the effect of

of the study was to obtain the favorable ratio between compound the pre-incubation of the amine 1b and paraformaldehyde leading

4a and the byproduct 4a’, which was identified by HPLC/MS as the to the first mechanism intermediate imine was evaluated. Thus, the

amide resulting from the direct coupling between protected protected hydroxylamine 1b and paraformaldehyde were stirred

hydroxylamine 1b and carboxymethyl nucleobase 2a.

during 1 h at room temperature in MeOH or isopropanol, the

carboxymethyl nucleobase 2a and isonitrile 3a were then added

Table 1 Optimization of reaction conditions for the synthesis of PNA and the mixture was stirred at rt for 24 h (entry 5 and 6, Table 1). In

monomer model 4a.

theses conditions, the formation of the byproduct 4a’ was slightly

reduced.

However, when the reaction was stirred at 60 °C for 10 h (entry 7,

Table 1), the compound 4a formation was improved from 23 to 35%

yield and the formation of the byproduct 4a’ was drastically

reduced from 36 to 12% yield.

Because the use of energy-efficient microwave heating to facilitate

chemical reactions often results in a dramatic acceleration of

reactions, resulting in cleaner outcomes and increased yields, the

effect of microwave irradiation (MWI) was evaluated at the same

temperature of 60 °C. Thus, the protected hydroxylamine 1b and

Entry

Solvent

T (°C)

Time

Pre-

incubation

NO

MWI

Yield

4a

Yield

4a’

1

2

MeOH

rt

24h

NO

YES

26%

5%

6%

paraformaldehyde were pre-incubated during

1

h, the

Isopropanol

MeOH

NO

2%

carboxymethyl nucleobase 2a and isonitrile 3a were then added

and the mixture was irradiated at 60 °C for 3 h. The reaction was

faster and no longer changed after 3 h. The compound 4a was

obtained with 45% yield and the byproduct 4a’ formation was

negligible (entry 8, Table 1). Under MWI, the rate of the reaction is

governed by the law of Arrhenius. The reaction time is halved each

time the temperature is raised of 10 °C, from 3 h reaction at 60 °C

to 1 min at 130 °C. To evaluate the thermal effect of MWI, reaction

was carried out at 70 °C for 1h30, 80 °C for 45 min, 100 °C for 15

min and 130 °C for 1 min (entry 9 to 12, Table 1). In all conditions,

the compound 4a was obtained with modest yields (30 to 40%) but

a substantial proportion (14 to 25%) of byproduct 4a’ was also

observed (14 to 25%). Therefore, if MWI has shown its ability to

reduce the reaction time, up to 1000 times when the reaction was

carried out at 130 °C and its ability to modestly improve the yield,

3

rt

48h

NO

12%

23%

19%

15%

35%

45%

40%

36%

33%

30%

4%

4

60

rt

10h

NO

36%

2%

5

24h

YES

6

Isopropanol

rt

24h

YES

Trace

12%

4%

7

60

70

80

100

130

10h

YES

8

3h

YES

9

1h30

45min

15min

1min

YES

14%

15%

20%

25%

10

11

12

YES

Reaction conditions: 1b (0.3 mmol), 2a (0.3 mmol), 3a (0.3 mmol), paraformaldehyde

(0.3 mmol). Yields are expressed in percentage of byproduct 4a’ formation followed by

HPLC and percentage of recovery of expected compound 4a.

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however the criterion of minimizing the formation of the byproduct 13, 14, Table 2), however, the yields remain similar or better than

4a’ was not fulfilled when temperatures above 60 °C were used.

those described by W. Maison^{11, 12}

.

Optimal conditions should be a compromise between reaction time, For 4f-h series (entries 6, 7, 8, Table 2) only monomers bearing

yield and purity of the compound 4a. Thus, MWI at 60 °C for 3 h in pyrimidine nucleobases were synthesized and good yields were

isopropanol preceded by a pre-incubation of the hydroxylamine 1b observed (50-60%). For the 4i-l series (entries 9, 10, 11, 12, Table 2),

and paraformaldehyde during 1 h seems to satisfy (entry 8, Table slightly lower yields were obtained, due to the slight decomposition

1).

of protected amine 1a in the reaction conditions.

To explore the generality and the substrate scope of the developed

condensation, several protected amines (1a-d), carboxymethyl

nucleobases (2a-e) and isonitriles (3a-c) were used. The

corresponding Ugi products (4a – 4n) were obtained in good yields

(Table 2) and purity higher than 95%, after purification by silica gel

column chromatography. Compounds (4a – 4n) were characterized

by ¹H and ¹³C NMR spectroscopy and HRMS.

To be used in the second Ugi-4CC reaction as building blocks, the

monomers 4a, 4c, 4d and 4e were first deprotected at their C-

terminus. To remove Boc protection, monomers were treated with

standard 50% TFA/DCM solution at 0 °C during 1h. After treatment

scavenger resin was added to quench the trifluoroacetate ions

generated during Boc removal. After filtration and evaporation,

deprotected PNA monomers 5a, 5c, 5d and 5e were obtained as

white powders, respectively, with 84, 81, 85 and 69% yield.

Table 2 Synthesis of PNA monomers 4a to 4n.

Dimer synthesis

To probe the strategy to synthesize PNA dimers, the Ugi product 6a

(GC PNA dimer model) served our exploration to study the effect of

solvent, reaction time and temperature under MW irradiation. We

mixed deprotected PNA monomer 5a, carboxymethyl nucleobase

2b, paraformaldehyde and isocyanide 3b and modulated

experimental conditions. Results are gathered in Table 3.

Entry

1

R

P

4a

4b

4c

4d

4e

4f

X

R

B

G^iBu

C^tBuBz

A^Bz

T

Yield

45%

40%

41%

65%

73%

51%

62%

54%

37%

40%

45%

41%

43%

1b, 2a, 3a

1b, 2b, 3a

1b, 2c, 3a

1b, 2d, 3a

1b, 2e, 3a

1b, 2b, 3b

1b, 2d, 3b

1b, 2e, 3b

1a, 2b, 3b

1a, 2c, 3b

1a, 2d, 3b

1a, 2e, 3b

1d, 2b, 3b

1b, 2a, 3c

OTBDPS

NHBoc

CH₂NHBoc

COOMe

2

Table 3 Optimization of reaction conditions for the synthesis of PNA

dimer model 6a.

3

4

5

U

6

C^tBuBz

7

4g

4h

4i

COOMe

T

8

COOMe

U

9

COOMe

C^tBuBz

A^Bz

T

Entry

Solvent

T (°C)

Time

Pre-

incubation

YES

MWI

Yield

6a

10

11

12

13

14

4j

NHBoc

COOMe

4k

4l

NHBoc

COOMe

1

2

3

4

5

6

7

MeOH

Isopropanol

DMF

rt

48h

NO

YES

7%

NHBoc

COOMe

U

4m

4n

NHMmt

OTBDPS

COOMe

C^tBuBz

G^iBu

YES

17%

19%

30%

39%

40%

43%

rt

48h

YES

CH₂NHFmoc

Isopropanol

DMF

60

70

100

3h

YES

Reaction conditions: Yields are expressed in percentage of recovery of expected

compound after purification by column chromatography. Conditions: 1 equi of each

reagent, pre-incubation, MWI, 60 °C, 3 h, isopropanol. R = reagent, P = product.

1h30

45 min

YES

Isopropanol

YES

The PNA monomers 4a-e and 4n were designed to be used in the

synthesis of PNA dimers where they will play the role of the amine

by their C-terminus in the second Ugi-4CC reaction. The monomers

4f-m were designed to be used in the synthesis of chimeric RNA-

PNA dimers.

Reaction conditions: 5a (0.1 mmol), 2b (0.1 mmol), 3b (0.1 mmol), paraformaldehyde

(0.1 mmol). Yields are expressed in percentage of recovery of expected compound 6a

after purification by column chromatography.

Using the standard Ugi-4CC conditions reactions, with 0.5 M

concentration of building blocks, in equimolar mixture, in MeOH,

isopropanol or DMF, at rt during 48 h, compound 6a was obtained

with 7%, 17% and 19% yield, respectively (entries 1, 2, 3, Table 3).

In all cases, a pre-incubation between deprotected PNA monomer

As indicated in Table 2 (entries 4, 5, 7, 8), monomers 4d, 4e, 4g and

4h, bearing pyrimidine nucleobases (thymine and uracil) were

obtained in excellent yields (54 to 73%), comparable or rather

higher to those described by P. Xu on similar PNA templates¹³

.

Moreover, for all products, the reaction conditions are improved

(less equivalent) and reaction time is much shorter (3h versus 48h).

A slightly decrease in the efficiency of the reaction was observed

with protected carboxymethyl nucleobases (entries 1, 2, 3, 6, 9, 10,

5a and paraformaldehyde during

1 h was performed as

recommanded for the synthesis of PNA monomers. The very low

yield obtained in MeOH was due to the unfavorable precipitation of

some building blocks, nevertheless the efficiency of the Ugi-4CC in

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these standard conditions is weak. As expected, the substitution of reaction conditions. To bypass this solubility bottleneck, one

MeOH by isopropanol or DMF affected positively the yield of the solution should be to add an ionic liquid to the reaction mixture to

reaction (entries 2 and 3, Table 3).

improve the phase transfer and strengthen the MWI effect.

The effect of the MWI was then evaluated. Thus, the deprotected

PNA monomer 5a and paraformaldehyde were stirred during 1 h at

room temperature in isopropanol, the carboxymethyl nucleobase Deprotection of dimers 6a-c and 6e

2b and isonitrile 3b were then added and the mixture was

irradiated at 60 °C for 3 h. The compound 6a was formed faster, the The PNA dimers 6a, 6b and 6c possess three types of protecting

reaction time was reduced 16 times and the yield was improved group. Among the different strategies of protection – deprotection

from 19 to 30% (entry 4, Table 3).

described in literature for the synthesis of PNA oligomers²¹, the

To evaluate the thermal effect of MWI, reaction was carried out at protections were chosen to be compatible with the synthesis of

70 °C for 1h30 and 100 °C for 45 min (entries 4, 5, 7, Table 3). In all chimeric RNA-PNA or PNA-RNA^{22, 23}and to avoid final deprotection

conditions, the yields are modest but serviceable (39 to 43%).

in strong acidic conditions. The nucleobases are protected by acyl

In conclusion, the synthesis of the dimer 6a by Ugi-4CC is inefficient groups and the C-terminus as a methyl ester function cleavable in

at rt, whatever the solvent used, due to the weak solubility of PNA alkaline conditions. The N-terminus is protected by a silyl group

monomer 5a and carboxymethyl nucleobase 2b. The use of MWI cleavable with fluoride anions²⁴

.

proved to be particularly beneficial, on the reaction time (60 times Our first deprotection strategy was to apply an unique treatment to

reduced) and on the efficiency of the reaction (6 times improved). cleave all protections in one step. A solution of NaOH (0.2 N or 5 N)

Thus, irradiation at 100 °C for 45 min in isopropanol preceded by a in MeOH or EtOH has been described^{25, 26}to perform acyl and silyl

pre-incubation of the deprotected PNA monomer 5a and group deprotection and ester hydrolysis. By using a solution of

paraformaldehyde seems to satisfy (entry 7, Table 3).

NaOH (0.2 N) in MeOH, we observed the rapid (< 1 h) saponification

of the methyl ester function, the partial deprotection of the acyl

To explore the generality and the substrate scope of the developed groups and the cleavage of the silyl group. An extended treatment

condensation, several PNA monomers (5a, 5c, 5d and 5e), of 16 h was required to observe complete deprotection of acyl

carboxymethyl nucleobases (2a, 2b, 2d and 2e), isonitrile (3b) and groups on purine nucleobases. However, this reaction time was

paraformaldehyde were used. The corresponding Ugi products (6a responsible of an important degradation of the PNA dimer by

– 6f) were obtained in moderate to good yields (Table 4) and purity cleavage of the amide bond between the nucleobase and the

higher than 95%, after purification by silica gel column acetamide backbone. Therefore, a strong alkaline treatment for an

chromatography. Compounds (6a – 6f) were characterized by ¹H unique deprotection, though attractive, is not possible.

and ¹³C NMR spectroscopy and HRMS.

Because aqueous ammonia solution is currently used for milder

nucleobases deprotection and solid support cleavage in

conventional oligonucleotide²⁷or PNA oligomers synthesis^{28, 29}, we

used alternatively NH₄OH solution (28%) in THF/MeOH at 55° C

(Scheme 2). After 6 h, complete deprotection of nucleobases and

ammonolysis of the ester function were observed. After

evaporation, TBAF (1M) solution in THF was added. Complete

deprotection of the silyl group was observed after 1 h. If the

treatment with TBAF solution was realized first, the methyl ester

function was partially hydrolyzed (40%) along with the silyl group

deprotection, leading to the formation of a mixture of one dimer

with acidic C-terminus and one with amide C-terminus after

ammoniacal treatment. The lack of orthogonality between

protecting groups imposes the order in which deprotections should

be performed Thus, the dimers 6a, 6b and 6c were solubilized in

MeOH / THF (1:3, v:v), a 28% NH₄OH solution was added and the

mixture was stirred 6 h at 55 °C in a sealed tube. After evaporation,

the residue was dissolved in THF and treated 1 h at rt with a

solution of 1M TBAF in THF to yield the PNA dimers 7a-c with 33, 23

and 32% yield respectively after reverse phase chromatography.

In order to generate a dimer with acidic C-terminus and because

thymine does not require nucleobase protection, dimer 6e was

deprotected using alternative procedure (Scheme 2). Compound 6e

was first treated with a solution of 1M TBAF in THF during 1 h at rt

to remove silyl group and then treated with NaOH 0.2 N in MeOH,

during 1 h at rt to hydrolyze the ester function, leading to

compound 7e with 45% yield after reverse phase chromatography.

Table 4 Synthesis of PNA dimers dimers 6a to 6f.

Entry

R

P

B₁

G^iBu

A^Bz

T

B₂

C^tBuBz

U

Yield

43%

35%

30%

25%

40%

30%

1

2

3

4

5

6

5a, 2b

5a, 2e

5c, 2b

5c, 2a

5d, 2d

5e, 2b

6a

6b

6c

6d

6e

6f

C^tBuBz

G^iBu

T

U

C^tBuBz

Reaction conditions: Yields are expressed in percentage of recovery of expected

compound after purification by column chromatography. Conditions: 1 equivalent of

each reagent, pre-incubation, MWI, 100 °C, 45 min, isopropanol. R = reagent, P =

product.

The PNA dimers 6a-f were designed to represent dimer sequences

bearing all purine and pyrimidine nucleobases. Yields vary between

25 and 43%. In comparison with the PNA monomer synthesis, a

decrease in the efficiency of the Ugi-4CC is observed and could be

explained by the lower solubility of PNA monomers 5a-e in the

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We describe below the complete NMR characterization of 4 PNA

dimers 7a-c and 7e in aqueous solution (For 1D ¹H and ¹³C NMR

spectra, see Experimental Section and Supporting Information S26-

S29) based on the major rotamers. To describe the dimers, we used

a nomenclature inspired from Brown et al.³⁵and commonly used by

others³⁶. The numbering inside the nucleobases B₁and B₂follows

Scheme 2 Synthesis of deprotected dimers 7a-c and 7e. Yields are the IUPAC recommendations defined for the nucleic acids (Figure

expressed in percentage of recovery of expected compound after 2).

purification by reverse phase column chromatography. Reagents

and Conditions: (a) 1) MeOH / THF (1:3, v:v), 28% NH₄OH, 55 °C, 6 h;

2) 1M TBAF in THF, rt, 1 h; (b) 1) 1M TBAF in THF, rt, 1 h; 2) NaOH

0.2N in MeOH, rt, 1 h.

NMR characterization of PNA dimers 7a-c and 7e in aqueous

solution

Nuclear Magnetic Resonance is among the analytical tools

systematically used to validate the structure of synthetic Fig. 2 Atom numbering and torsion angles convention in the PNA

compounds in a complementary way to other techniques such as dimers 7a-c, 7e and dimer model.

mass spectroscopy. However in the case of small PNA oligomers, its

1

use is limited by the complexity of the spectra. Indeed, for each The H and ¹³C assignments were obtained by a combined analysis

monomer of a PNA oligomer, the restricted rotation around the of J-coupling correlation spectroscopy (DQF-COSY³⁷), heteronuclear

amide bond linking the nucleobase to the acetamide backbone multiple bond (HMBC^{38, 39}) and single bond (HSQC⁴⁰) correlation

leads to the existence of two stable conformers^{30, 31}. The energetic experiments. In addition to the conformational equilibrium and its

barrier between them is high and the interconversion rate is often effects on the spectra, the structure of the PNA itself with its

slow enough (in the order of 1 sec^-1)³²to observe a separate data abundance in heteroatoms and quaternary carbons makes the

set for each conformer. The coexistence of several species in assignment more difficult than for natural oligonucleotides.

solution thus explains why rather few NMR data exists to describe Consequently and in order to discard any error, the identification of

short PNAs oligomers^{30, 33}. Indeed, lists of the ¹H and ¹³C chemical the nuclei was essentially based on a careful examination of the

shifts are usually given without assignment to the corresponding heteronuclear couplings. Through-space interactions were used to

nuclei, or with a reliable but partial assignment. A possibility to confirm the assignments and get conformational data and ROESY

simplify the analysis consists in accelerating the isomerization rate experiment⁴¹allowed to distinguish the dipolar interactions from

in order to approach the fast exchange regime and thus get a single the conformational exchange between the conformers since the

averaged data set. This strategy was applied by Plöger et al. to latters had opposite sign.

study fluor-containing short PNA oligomers in DMSO³⁴. If working at In a general way, the assignment process was done using as starting

elevated temperature effectively led to simplified spectra, point the aromatic protons of the nucleobases (H₈for the purines

improved but nevertheless still incomplete assignments were and H₆for the pyrimidines). For example for the dimer 7e, the

obtained. Moreover this method is limited by the possible resonances of the nuclei composing each thymidine were identified

alteration of the compounds. We thus investigated NMR study in from the correlations to the aromatic H₆protons (at 7.19 and 7.48

the conditions of the slow exchange regime occuring in D₂O.

ppm respectively), the C₄quaternary carbons (169.3 and 169.4

We first used variation of temperature experiments to emphasize ppm) being distinguished from C₂(154.8 ppm) according to their

1

the existence of the rotamers. The H NMR spectrum of compound correlation to the methyl groups. Each methylcarbonyl linker was

7e at 298 K (S29, Supporting Information) presented four sets of then connected to the corresponding nucleobase by the

signals attributed to the putative existence of the 4 conformers. correlations implying the methylene H_8'protons with the C₂and C₆

Several ¹H spectra acquired on a temperature range of 298 to 363 K (146.4 ppm) carbons, a third correlation allowing to assign the

(S30, Supporting Information) showed

a broadening of the carbonyl C_7'. On the pseudo-peptidic backbone, the two H_2'-H_3'spin

resonances line shapes and coalescence was reached around 360 K. systems of the aminoethyl moieties located on the COSY spectrum

Resulting in the acceleration of the exchange rate between the were differentiated from the higher chemical shift of the 2' protons

different conformations, this behavior confirmed the existence of (at 3.81 ppm) of the first monomeric unit deshielded by the

several conformers. In D₂O, the four PNA dimers 7a-c and 7e terminal hydroxyl group. The C_3'-H_5'and C_6'-H_5'correlations were

exhibited similar behaviors. The ¹H spectra revealed several data used to complete the peptidic chain assignment of each monomer.

sets of rather thin peaks, characteristic of slow exchanges at the From the correlations between the carbonyls C_7'and the

NMR time scale. In each case one of the conformers appeared to be methylenes H_3'and H_5'in the backbone, each fragment constituted

significantly predominant over the others (at more than 50 %) and of the base with its carboxymethyl linker were distinctly linked to

1

it was thus possible to completely assign both the H and ¹³C nuclei the peptidic sequence. Finally the acid terminal end was connected

of these major conformers.

to the rest of the molecule as shown by the correlations of the

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methylene H_10'with the two carbonyls C_6'and C_11'. The main NMR studies that the carbonyls of the base linkers are oriented

correlations observed for the dimer 7e are represented on Scheme toward the C-terminal extremity of the PNA strand^{35, 36}

3.

The rate of exchange was calculated as 0.5-2 s^-1at 37^oC³⁴. The

.

activation energy for the interconversion between cis- and trans-

conformers has been determined by variable-temperature 1H-NMR

spectroscopy on four PNA monomers as 19+-2 kcal/mol³⁴. This

energy barrier is apparently too high, that could be overcome on a

time scale of about one microsecond. Therefore, our MD

simulations were started from various initial conformers (see S62,

Supporting Information). While the secondary amide bond (i.e.

backbone torsion angle ζ) was always set to trans, for tertiary

amide bonds in linkages connecting bases to backbone (i. e. χ₁and

χ_1’torsion angles), all possible combinations of conformers (i.e. cis-

Scheme 3 Selected heteronuclear long-range correlations in the cis, cis-trans, trans-cis and trans-trans) were used leading to starting

dimer 7e. For a better clarity, most of the protons were not structures I-IV (see S62, Supporting Information). In fact, two four-

reported on the structure and some correlations were omitted.

membered sets of initial structures were constructed. In crystal

structures, backbone torsion angles β’/δ prefer synchronously

The ¹H spectrum at 323 K of dimer 7a revealed several data sets, either +gauche or -gauche conformers. The +gauche conformers

one of the conformers prevailing significantly at 62%. The are usually found in crystal structures of right-handed homo- or

resonances of all the non exchanging protons were assigned. For heteroduplexes that contained at least one PNA strand^{35, 43}. In

the ¹³C nuclei, only the C₂and C₆of the guanine were not contrast, the ideal value for β’/δ in left-handed PNA duplexes is -

determined. The ¹H spectrum of dimer 7b revealed also a major 60^o36. Therefore, both conformers (i.e. +/-gauche) were initially set

conformer (50 %), for which all the protons resonances were for β’/ δ in our simulated systems leading to two four-membered

assigned. The quaternary carbons of the bases were observed with sets of initial structures (i.e. I-IVa vs. I-IVb - see S62, Supporting

difficulty, and exhibited particularly broad signals while C₈was Information). Microsecond MD simulations were produced using

completely missing on the 1D ¹³C NMR spectrum. Its chemical shift programmable GPUs, which is a big step forward considering that

was nevertheless deduced from its correlation to H_8’visible on the previous calculations on PNA were mere minimizations of either

HMBC spectrum. No long range correlation was detectable for the molecular-mechanical energy of helical structures⁴⁴or quantum-

carbons C₂and C₆of the guanine and the two corresponding mechanical energy of dinuleotides⁴⁵or at best short 100 ps MD

resonances at 161.3 and 156.6 ppm were thus not individually simulations of CT-dimer³²or 1.5 ns MD simulations of PNA single

assigned. The dimer 7c at 298 K was found to exist as a mixture of 4 strands⁴⁶

Many inter-conversions of torsion angles β’/δ between +gauche/-

.

slowly interconverting conformers in a ratio of 67%, 17%, 9% and

8% as determined from the integration of the isolated H₆protons of gauche conformers were observed in our MD trajectories (see S61,

the cytidine. All the resonances were individually assigned. The ¹³C

Supporting Information). Many other torsion angles in PNA

resonances were determined from the collected HMBC experiment

backbone (i.e. δ, ε, α’, β', γ‘) or in backbone-base linkages (i.e. χ₂,

χ₃, χ_2’, χ_3’) were also conformationally fluidic, which is usually

data. For dimer 7e, the respective population of the 4 conformers

was estimated as 58%, 24%, 12% and 6% according to the

considered as an asset with respect to binding of PNA to

integration of the H₆signals (S30, Supporting Information). The

complementary DNA/RNA strand. Here, it caused even a complete

complete assignment of the major conformer was achieved, and

de-stacking of dimers that was more or less reversible (see time

the ¹H resonances were also determined for the second

evolutions of either N₃/N₁or C₈/C₆inter-atomic distances in S61,

predominant one.

Supporting Informations). In MD simulations Ia, Ib, IIa, IIb (i.e. for

The whole of the chemical shifts (¹H and ¹³C) for the four dimers are

PNA dimers with either cis-cis or cis-trans conformers in terms of

reported in the Experimental Section. These NMR data taken

the χ₁/χ_1’torsion angles), the base-stacked ordered structure

together with the mass spectrometry analysis, confirmed the

appeared over long periods throughout MD trajectories lasting for

structure of the dimers 7a-c and 7e.

900-1.200 ns. In MD simulations IIIa, IIIb (i.e. for the trans-cis

conformers in terms of the χ₁/χ_1’torsion angles), destacked

conformers prevailed. Finally, in MD simulations IVa, IVb (i.e. for the

trans-trans conformers of the χ₁/χ_1’torsion angles) stacking was

observed only rarely. Taken together, relative viability of base

stacking could at least partially explain uneven abundance of PNA

conformers observed in our NMR experiments. Representative

conformers of cis-cis and cis-trans PNA structures with the most

viable base-stacking in MD simulations Ia, IIa are depicted in Figure

3. The cis-cis PNA structure (see Figure 3A) is one that is usually

found in crystal structures of either duplexes or triplexes including

Conformational preference of the tertiary amide bond in GC PNA

dimer model by MD simulations

The PNA monomers were extensively described in the literature

and many examples report that among the 2 main conformers

resulting of the cis-trans equilibrium around the methylcarbonyl

linker amide bond, the cis form (that is to say, the one

corresponding to the C_7’carbonyl pointing towards the glycine

32, 42

moiety) significantly (70:30) predominates^31,

. The cis

conformational preference becomes even more pronounced in PNA

duplexes, where it’s now well established from crystallography and

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Journal Name

at least one PNA strand. The cis-cis conformation puts the PNA C_7'

carbonyl groups in positions isosteric to an RNA C_2', facilitating

maximal solvent exposure of the two carbonyl oxygens. This

arrangement appears to be optimal if one considers polarizations of

individual atoms, as it puts into a close spatial proximity the oxygen

atom from the carbonyl group of the residue with guanine base and

the hydrogen atom on the secondary nitrogen along the backbone.

A

B

In references⁴⁴

, the “inter-residue” hydrogen bonding stabilizing

PNA-DNA and PNA-RNA hybrids was proposed on the base of

molecular mechanics calculations produced in the gas phase.

Nevertheless, it was not found neither in crystal structures of helical

complexes nor in short (100 ps) MD simulations produced for a CT-

dimer in a cube of water³²and this hydrogen bonding was not

stabilized even in the course of our microsecond MD runs. In MD

simulation Ia (i.e. starting from the left-hand cis-cis conformer) we

cis-cis conformer

cis-trans conformer

Fig. 3 Representative conformers of GC PNA dimer model with the

most viable base-stacking in MD simulations. a) MD run 1a (t = 800

'

ns): torsion angle

β

' in -gauche,

χ

in cis,

χ

in cis. The cis-cis

conformation puts the PNA C_7'carbonyl group in a position isosteric

to an RNA C_2', facilitating maximal solvent exposure of the two

observed fluctuations for β’, however, δ was stable. This means that

we did not observe a complete transition from the left- to right-

handed arrangement. Nevertheless, transition in the opposite

direction was found in the MD simulations IIa. It could indicate, that

PNA tends to be pre-organized in the left-handed arrangement

(which is well-known from PNA:PNA duplexes) and the right-handed

arrangement is imposed on PNA by DNA/RNA counterparts.

According to our best knowledge, the cis-trans PNA structure (see

Figure 3B) has not yet been studied. This conformer is potently

stabilized by the intra-molecular hydrogen bond established

between the secondary backbone amidic proton and the C_7'

carbonyl group from the monomer with cytosine base (see Figure

3B, and S61, Supporting Information). Earlier energy-minimized

structures of Watson-Crick base-paired decameric duplexes of PNA

with A-, B- and Z-DNA and A-RNA indicated that such “intra-

carbonyl oxygens. b) MD run IIa (t = 600 ns): torsion angle

gauche, in cis,

^'in trans.

β' in -

χ

Validation of the conformational preference of the tertiary amide

bond in dimers 7a and 7e by NMR studies

The conformational preference around the N₄-C_7’amide bonds can

be established from the ROESY spectrum, using the correlations of

the carboxymethyl linker protons H_8’with the methylenes of the

backbone^{32, 47}. ROESY spectra were thus acquired for 7a and 7e

(Figure 4A and 4B respectively). For both compounds, the two H_8’

groups were found to have strongest cross correlations with the

protons of the ethylenediamino moiety H_3’, suggesting the C_7’

carbonyl groups to be turned towards the C-terminal extremity (cis-

cis conformation). Among studied dimers, the dimer 7e is

particularly interesting because the splitting of the resonances and

the respective population of the 4 conformers made possible to

assign the ¹H resonances of the second majoritary conformer as

well. In this second conformer, the strong H_8’-H_3’correlation was

found for the N-terminal residue (T₁) while the conformation

around the amide bond of the C-terminal residue was determined

as trans, with the H₈’ methylene oriented to the glycine, as deduced

form the H_8’-H_5’correlation (Figure 4B). The populations of the two

minor conformers were too weak to get reliable NOE data. The

result observed at the dimeric stage is in agreement with the

monomeric behavior.

residue” hydrogen bonding does not contribute to their stability⁴⁴

.

However, these oligonucleotides were rather in the “all-trans”

arrangement, that doesn’t lead to viable base stacking in our MD

simulations IVab (see Figure S61, Supporting Information). On the

other hand, the cis-trans PNA could be destabilized by unfavorable

arrangement of oxygens from both carbonyl groups which are

directed each against the other. It must lead to a substantial

repulsion. Maybe it opens up space for the stabilization of this

conformer by replacing the carbonyl group in the monomer with

the guanine base. Compatibility of cis-cis and cis-trans PNA

structures with the active site of RdRp will be subject to further

examination.

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A

7a: B₁=G, B₂= C, R = NH₂

7e: B₁=B₂=T, R = OH

B

Fig. 4 (Left) Selected region of the ROESY spectra of 7a (A) and 7e (B). (Right) Favoured orientations of the N_4’-C_7’bonds determined from

the throught-space correlations observed between H8’ and the backbone’s methylenes for the major conformer (in red) and second major

conformer (for 7e only).

Finally, from the chemical shifts collected for all the dimers (7a-e as

well as the protected ones 6a and 6c) it appeared that among the

Conclusions

two H8’ methylenes of the nucleobase linkers, the one at the C

Peptide Nucleic Acids have received great attention as tools in

molecular biology and biotechnology. Many analogues of PNA were

designed to improve their physico-chemical and biological

properties. Faster and more efficient methodologies for their

synthesis need to be explored to circumvent the limitations of the

classical peptide chemistry. This work describes an efficient, rapid

and attractive solution phase strategy to obtain Peptide Nucleic

Acid monomers and dimers by using microwave-promoted Ugi

multicomponent reactions. Combining these two versatile tools is a

particularly profitable method in current organic synthesis. Hence,

we generated efficiently 14 PNA monomers (4a to 4n, 37 to 73%

isolated yield) by using one Ugi-4CC reaction and 6 PNA dimers (6a

to 6f, 25 to 43% isolated yield) by using two consecutive Ugi-4CC

terminal extremity was systematically found at a lower value. The

effect was found to be more important when the N terminal

extremity was bearing a purine as nucleobase (-0.4 to -1.0 ppm)

rather than a pyrimidine (-0.2 ppm). This shield can be explained by

the presence of this C-terminal H8’ in the vicinity of the magnetic

anisotropy cone of the N-terminal nucleobase and is consistent with

the structures obtained for the GC model (vide supra) and for the TT

dimer⁴⁵

.

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reactions under microwave irradiation. This method is particularly recorded with

Journal Name

a

Q-TRAP mass analyzer under electrospray

attractive in terms of molecular diversity, simplicity and atom ionization (ESI) in positive or negative ionization mode detection.

economy along with using readily available building blocks. High-resolution mass spectra (HRMS) were recorded with a Q-Tof

Although the PNA dimers exist in solution as 4 conformational mass analyzer under electrospray ionization (ESI) in positive or

rotamers, we have shown that it was possible to assign the negative ionization mode detection. LC/MS spectra were recorded

resonances of the major species and thus to get a complete data on an Accela 600 thermofisher (HPLC) with an Hypersil-Gold C18

sets for both ¹H and ¹³C nuclei. MD simulations were run (2.5 μm, 2.1x50 mm) column and Finnigan survey MSQ (MS) under

independently on a GC dimer model and established the supremacy electrospray ionization (ESI) in positive and negative ionization

of the cis-cis conformer, as result that was experimentally mode detection. Compounds were eluted with a gradient of a

confirmed by NMR.

solution of 2 to 100% of 0.01% formic acid in ACN solution with a

550 μL/min flow rate. High-pressure liquid chromatography (HPLC)

spectra were recorded on a Waters apparatus using a Waters 600

pump and a Waters 916 photodiode array detector with an Xbridge

C18 (10 μm, 4.6x150 mm) column provided with a pre-column

Xbridge (3.5 μm, 100 Å, 4.6x20 mm). Compounds were eluted using

a linear gradient of 10 to 100% of ACN or using a gradient of 0 to

Experimental

General Information

TLC was performed on Merck silica coated plates 60F₂₅₄

.

Compounds were revealed on UV light (254 nm). Column 100% of 0.05M triethylammonium bicarbonate buffer (pH 7.5) in

chromatography was performed on silica gel Merck G60 230-240 50% ACN with a 1mL/min flow rate.

mesh. Flash chromatography was performed on a Biotage SP4 using

silica gel column with an elution profile adapted to the crude. All (2-isocyano-ethyl)-carbamic Acid tert-butyl Ester (3a). tert-butyl N-

moist-sensitive reactions were carried out under anhydrous (2-aminoethyl)carbamate 1a (3.8 g, 23.70 mmol) was dissolved in

conditions using dry glassware, anhydrous solvents and argon ethyl formate (20 mL) and the solution was heated at reflux for 18

atmosphere. All anhydrous solvents were purchased in Aldrich. h. Upon cooling to room temperature, the reaction mixture was

Microwave-promoted syntheses were performed on a Biotage^®concentrated under reduced pressure to furnish the corresponding

initiator using sealed vessels and the reaction temperature was formamide 1’a (4.4 g, quantitative yield). R_f0.55 (DCM/MeOH, 9/1);

monitored using an external IR sensor.

¹H NMR (250 MHz, CDCl₃) δ (ppm) 8.12 (s, 1H, COH), 6.36 (s, 1H,

The NMR experiments were acquired on Bruker spectrometers NH), 4.89 (s, 1H, NH), 3.40-3.28 (m, 2H, CH₂), 3.26-3.13 (m, 2H, CH₂),

equipped of 14.1 and 9.4 T magnets (AVANCE III 600 and AVANCE III 1.37 (s, 9H, CH₃). ¹³C NMR (63 MHz, CDCl₃) δ (ppm) 161.7 (CO),

HD 400 NMR systems). The samples were prepared by directly 156.9 (CO), 79.9 (C-tBu, C), 40.3 (CH₂), 39.4 (CH₂), 28.3 (CH₃). TOF

dissolving the products into deuterated solvents purchased from MS ESI⁺189.1 [M+H] ⁺.

Eurisotop (Saint Aubin, France). The concentrations of the dimers

were ranging from 15 to 27 mM for analysis in 5 mm diameter A solution of formamide 1’a (4.4 g, 23.30 mmol, 1.0 equiv.) and

tubes. NMR microtubes with System Match (Bruker) were used for triethylamine (15.7 mL, 116.50 mmol, 5.0 equiv.) in anhydrous THF

the products available in smaller quantities (dimers 7b and 7c) in (72 mL) was cooled to 0°C, and phosphorous oxychloride (2.4 mL,

order to reach concentrations of 70 and 8.3 mM respectively. The 25.60 mmol, 1.1 equiv.) was added dropwise. The reaction mixture

NMR data of 7c and 7d were collected at 298 K and the less soluble was stirred at 0 °C for 2 h and at room temperature for 1 h. The

7a and 7b were studied at 323 K.

reaction mixture was then poured into cold H₂O (2 x reaction

In organic solution, the spectra were internally referenced volume), the organic layer was separated, and the aqueous layer

according to the solvent residual peak (in DMSO: δ¹³C 39.5 ppm; was extracted with Et₂O (3 x 100 mL). The combined organic

δ¹H 2.50 ppm; in CDCl₃: δ¹³C 77.2 ppm; δ¹H 7.26 ppm; in CD₃OD: fractions were washed with brine, dried over Na₂SO₄filtered and

δ¹³C 49.0 ppm; δ¹H 3.31 ppm). In aqueous medium, the DSS concentrated under reduced pressure. The crude was purified over

1

resonance was used to calibrate the H spectra at 0 ppm while the silica gel column chromatography (1:1 EtOAc – hexane) to give (2-

¹³C spectra were indirectly referenced with

Ξ . The isocyano-ethyl)-carbamic acid tert-butyl ester 3a as a white powder

_C=25.144953⁴⁸

1

2D spectra were obtained from standard pulse sequences with 1K (2.83 g, 71% yield). H NMR (250 MHz, CDCl₃) δ (ppm) 4.91 (s, 1H,

to 2K data points in F2 and 256 to 512 data points in F1. Phase- NH), 3.46 (t, 2H, J = 5.3 Hz, CH₂), 3.37-3.25 (m, 2H, CH₂), 1.39 (s, 9H,

sensitive ROESY spectra were carried out with a mixing time of 300 CH₃). ¹³C NMR (63 MHz, CDCl₃) δ (ppm) 157.6 (CN), 155.7 (CO), 80.3

ms. Multiplicity-edited HSQC experiments using echo-antiecho were (C-tBu, C), 42.1 (CH₂), 39.9 (CH₂), 28.4 (CH₃). LC/MS ESI⁺171.15

1

acquired with an evolution delay optimized for J_CH=145 Hz. [M+H]⁺; 340.89 [2M+H]⁺.

Magnitude mode ¹H-¹³C HMBC spectra were acquired with an

evolution period optimized for an average long-range coupling of (2-isocyano-ethyl)-carbamic acid 9H-fluoren-9-yl-methyl ester (3c).

8.5 Hz.

N-(2-aminoethyl)carbamic acid 9H-Fluoren-9-ylmethyl ester

Chemical shifts are given in parts per million (ppm) and coupling hydrochloride 1c (501 mg, 1.57 mmol, 1.0 equiv.) was dissolved in

constants are given in hertz. Signal splitting patterns are described ethyl formate (9 mL) and the solution was heated at reflux and

as singlet (s), doublet (d), triplet (t), quartet (q), broad signal (bs), triethylamine was added (255 μL, 1.89 mmol, 1.2 equiv.). The

doublet of doublet (dd), doublet of triplet (dt), doublet of quartet solution was stirred at reflux for 18 h. Upon cooling to room

(dq), and multiplet (m). Low-resolution mass spectra (LRMS) were temperature, the reaction mixture was concentrated under reduced

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pressure and the crude was purified over silica gel column J= 6.1 Hz, H_2’), 3.62 (t, 2H, rotamer 2, J= 5.4 Hz, H_3’), 3.47 (t, 2H,

chromatography (DCM - MeOH) to furnish the corresponding rotamer 1, J= 6.1 Hz, H_3’), 3.13 (m, 2H, rotamer 1, H_10’), 3.04 (m, 2H,

formamide 1’c (519 mg, 94% yield). ¹H NMR (250 MHz, CDCl₃) δ rotamer 2, H_10’), 3.04 (m, 2H, rotamer 1, H_11’), 2.94 (m, 2H, rotamer

(ppm) 7.94 (s, 1H, COH), 7.82 (d, 2H, J = 7.2 Hz, H arom), 7.61 (d, 2H, 2, H_11’), 2.75 (sept, 1H, J= 6.9 Hz, iBu-CH), 1.36 (s, 9H, tBu-Boc), 1.10

J = 7.2 Hz, H arom), 7.30 (m, 4H, H arom), 4.24 (d, 2H, J=6.6 Hz, (d, 6H, J=6.9 Hz, iBu-CH₃), 1.03 (s, 9H, tBu-TBDPS). ¹³C NMR (100

CH₂), 4.14 (t, 1H, J=6.6 Hz, CH), 3.15-2.90 (m, 4H, 2CH₂). ¹³C NMR MHz, DMSO-d₆): δ (ppm) 180.1 (C-iBu, CO), 167.9 (C_6’, CO), 167.1

(63 MHz, CDCl₃) δ (ppm) 161.2 (CO), 156.1 (CO), 143.9 (C), 140.7 (C), (C_7’, CO, rotamer 1), 166.4 (C_7’, CO, rotamer 2), 155.7 (C-Boc, CO,

127.6 (CH arom), 127.0 (CH arom), 125.1 (CH arom), 120.0 (CH rotamer 1), 155.6 (C-Boc, CO, rotamer 2), 154.8 (C₆, CO), 149.3 (C₄,

arom), 65.3 (CH₂), 46.7 (CH), 40.4 (CH₂), 39.9 (CH₂). LC/MS ESI⁺C, rotamer 1), 149.2 (C₄, C, rotamer 2), 147.8 (C₂, C), 140.5 (C₈, CH,

310.91 [M+H]⁺; 621.12 [2M+H]⁺.

rotamer 1), 140.0 (C₈, CH, rotamer 2), 135.0 (C_ortho-TBDPS, CH),

132.8 (C_ph-TBDPS, C, rotamer 1), 132.6 (C_ph-TBDPS, C, rotamer 2),

A solution of formamide 1’c (500 mg, 1.61 mmol, 1.0 equiv.) and 130.0 (C_para-TBDPS, CH, rotamer 2), 129.9 (C_para-TBDPS, CH, rotamer

triethylamine (1.0 mL, 8.06 mmol, 5.0 equiv.) in anhydrous DCM (4 1), 128.0 (C_meta-TBDPS, CH, rotamer 2), 127.9 (C_meta-TBDPS, CH,

mL) was cooled to 0 °C, and phosphorous oxychloride (165 μL, 1.77 rotamer 1), 119.5 (C₅, C), 77.7 (CtBu-Boc, C), 61.7 (C_2’, CH₂, rotamer

mmol, 1.1 equiv.) was added dropwise. The reaction mixture was 2) 60.8 (C_2’, CH₂, rotamer1), 50.6 (C_5’, CH₂, rotamer1) 48.4 (C_5’, CH₂,

stirred at 0 °C for 2 h and at room temperature for 1 h. The reaction rotamer2), 49.3 (C_3’, CH₂, rotamer 1) 49.2 (C_3’, CH₂, rotamer2), 44.2

mixture was then poured into cold H₂O (2 x reaction volume), the (C_8’, CH_2,rotamer 2), 44.1 (C_8’, CH_2,rotamer1), 39.4 (C_11’, CH₂), 38.7

organic layer was separated, and the aqueous layer was extracted 3 (C_10’, CH₂), 34.6 (C-iBu, CH), 28.2 (C-tBu-Boc, 3CH₃), 26.7 (C-tBu-

times with DCM. The combined organic fractions were washed with TBDPS, 3CH₃,rotamer 2), 26.6 (C-tBu-TBDPS, 3CH₃, rotamer 1), 18.8

brine, dried over Na₂SO₄filtered and concentrated under reduced (C-iBu, 2CH₃). HRMS ESI⁺calcd for C₃₈H₅₃N₈O₇Si⁺(M+H)⁺761.3806,

pressure. The crude isonitrile was purified over silica gel column found 761.3799.

chromatography (1:1 EtOAc – hexane) to give (2-isocyano-ethyl)-

carbamic acid 9H-fluoren-9-yl-methyl ester 3c as a white powder 2-[(((N⁴-(4-tert-butylbenzoyl)cytosin-1-yl)acetyl)-(2-(tert-

1

(194 mg, 41% yield). H NMR (250 MHz, CDCl₃) δ (ppm) 7.70 (d, 2H, butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-(tert-butyloxycarbonyl-

J = 7.2 Hz, H arom), 7.51 (d, 2H, J = 7.3 Hz, H arom), 7.39-7.12 (m, amino)ethyl]acetamide (4b).

4H, H arom), 4.37 (d, 2H, J = 6.6 Hz, CH₂), 4.15 (t, 1H, J = 6.6 Hz, NH), A mixture of 2-{[tert-butyl(diphenyl)silyl]oxy}ethanamine 1b (200

3.41 (m, 4H, CH₂). ¹³C NMR (63 MHz, CDCl₃) δ (ppm) 155.2 (CO), mg, 0.67 mmol, 1.0 equiv.) and paraformaldehyde (20 mg, 0.67

142.7 (C), 140.4 (C), 126.8 (C arom), 126.1 (C arom), 123.9 (C arom), mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 1.3 mL) was

119.0 (C arom), 66.0 (CH₂), 46.2 (CH), 40.9 (CH₂), 39.3 (CH₂). LC/MS placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. (2-

ESI⁺292.93 [M+H]⁺.

Isocyano-ethyl)-carbamic Acid tert-butyl Ester 3a (113.7 mg, 0.67

mmol, 1.0 equiv.) and

N⁴-(4-tert-butylbenzoyl)-1-

Preparation of compounds 4a-n.

(carboxymethyl)cytosine 2b (220.2 mg, 0.67 mmol, 1.0 equiv.) were

then added dropwise. The resulting solution was irradiated at 50 W

(60 °C) for 3 h. The reaction mixture was concentrated to dryness

under reduced pressure and the residue was purified by column

chromatography (DCM with 5% MeOH) to afford 4b as a white

2-[(((N²-(isobutanoyl)guanin-9-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-(tert-butyloxycarbonyl-

amino)ethyl]acetamide (4a).

A mixture of 2-{[tert-butyl(diphenyl)silyl]oxy}ethanamine 1b (500 powder (273.8 mg, 50% yield). Tr-HPLC: 26.9 min (> 99%, 10-100%

1

mg, 1.67 mmol, 1.0 equiv.) and paraformaldehyde (50.2 mg, 1.67 ACN in 40 min). H NMR (400 MHz, DMSO-d₆) δ (ppm) 11.1 (s, 1H,

mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 3.3 mL) was NH-tBuBz), 8.20 (t, 1H, rotamer 1, J=5.8 Hz, H_9’), 7.83 (t, 1H,

placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. (2- rotamer 2, J= 5.8, H_9’), 7.98-7.97 (m, 2H, H_ortho-tBuBz), 7.83-7.80 (m,

Isocyano-ethyl)-carbamic acid tert-butyl ester 3a (284.3 mg, 1.67 1H, H₆), 7.65-7.61 (m, 4H, H_ortho-TBDPS), 7.54-7.52 (m, 2H, H_meta

-

mmol, 1.0 equiv.) and N²-(isobutanoyl)-9-(carboxymethyl) guanine tBuBz), 7.46-7.44 (m, 6H, H_meta/para-TBDPS), 7.32-7.29 (m, 1H, H₅),

2a (466.5 mg, 1.67 mmol, 1.0 equiv.) were then added dropwise. 6.85 (t, 1H, rotamer 1, J= 5.8 Hz, H_12’), 6.77 (t, 1H, rotamer 2, J= 5.8

The resulting solution was irradiated at 50 W (60 °C) for 3 h. The Hz, H_12’), 4.68 (s, 2H, H_8’), 4.17 (s, 2H, rotamer 1, H_5’), 3.94 (s, 2H,

precipitate was filtered, washed with cold MeOH to give 4a as a rotamer 2, H_5’), 3.86 (t, 2H, rotamer 1, J= 5.8 Hz, H_2’), 3.70 (t, 2H,

white powder (563 mg, 45% yield). Tr-HPLC: 23.6 min (> 95%, 10- rotamer 2, J= 5.8 Hz, H_2’), 3.59 (t, 2H, rotamer 1, J= 5.8 Hz, H_3’), 3.49

1

100% ACN in 40 min). H NMR (400 MHz, DMSO-d₆) δ (ppm) 12.07 (t, 2H, rotamer 2, J= 5.8 Hz, H_3’), 3.14 (m, 2H , rotamer 1, H_10’), 3.05

and 12.06 (bs, 1H, H₁), 11.69 and 11.65 (bs, 1H, NH-iBu), 8.26 (t, 1H, (m, 2H, rotamer 2, H_10’), 3.04 (m, 2H, rotamer 1, H_11’), 2.97 (m, 2H,

rotamer 1, J=5.4 Hz, H_9’), 7.94 (t, 1H, rotamer 2, J=5.2 Hz, H_9’), 7.77 rotamer 2, H_11’), 1.37 (s, 9H, tBu-Boc), 1.31 (s, 9H, tBuBz), 1.01 (d,

(s, 1H, rotamer 1, H₈), 7.47 (s, 1H, rotamer 2, H₈), 7.64 (d, 2H, H_ortho

-

9H, tBu-TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 168.4 (C_7’,

TBDPS, rotamer 2), 7.59 (d, 2H, H_ortho-TBDPS, rotamer 1), 7.46-7.42 CO), 168.1 (C_6’, CO), 167.6 (C₂, CO), 163.8 (C₄, C), 156.1 (C-Boc, CO),

(m, 6H, H_meta/para-TBDPS), 6.85 (t, 1H, rotamer 1, J=5.4 Hz, H_12’), 6.74 155.6 (C-tBuBz, CO), 155.6 (C_para-tBuBz, C), 151.6 and 151.3 (C₆,

(t, 1H, rotamer 2, J=5.4 Hz, H_12’), 5.02 (s, 2H, rotamer 2, H_8’), 4.97 (s, rotamers, CH), 135.5 (C_ortho-TBDPS, CH), 133.3 (C_ph-TBDPS, C), 130.9

2H, rotamer 1, H_8’), 4.17 (s, 2H, rotamer 1, H_5’), 3.94 (s, 2H, rotamer (C_ph-tBuBz, C), 130.3 (C_para-TBDPS, CH), 128.8 (C_meta-tBuBz, CH),

2, H_5’), 3.88 (t, 2H, rotamer 2, J= 5.4 Hz, H_2’), 3.68 (t, 2H, rotamer 1, 128.4 (C_meta-TBDPS, CH), 125.7 (C_ortho-tBuBz, CH), 96.2 (C₅), 78.1 (C-

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tBu-Boc, C), 62.4 and 61.6 (C_2’, rotamers, CH₂), 51.5 and 49.9 (C_5’, irradiated at 50 W (60 °C) for 3 h. The reaction mixture was

rotamers, CH₂), 50.2 and 50.0 (C_3’, rotamers, CH₂), 39.5 and 29.8 concentrated to dryness under reduced pressure and the residue

(C_11’, rotamers, CH₂), 35.3 (C-tBu-tBuBz, C), 31.3 (C-tBu-tBuBz, CH₃), was purified by column chromatography (DCM with 5% MeOH) to

28.3 (C-tBu-Boc, CH₃), 27.1 (C-tBu-TBDPS, CH₃), 19.1 (C-tBu-TBDPS, afford 4d as a white powder (217.6 mg, 65% yield). Tr-HPLC: 23.1

C). HRMS ESI⁺calcd for C₄₄H₅₉N₆O₇Si⁺(M+H)⁺811.4214, found min (> 98%, 10-100% ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆):

811.4208.

δ (ppm) 11.31 (1H, s, H₃), 8.19 (t, 1H, rotamer 1, J= 5.8 Hz, H_9’), 7.88

(t, 1H, rotamer 2, J= 5.8 Hz, H_9’), 7.67-7.62 (m, 4H, H_ortho-TBDPS),

7.53 – 7.44 (m, 6H, H_meta/para-TBDPS), 7.27 (s, 1H, rotamer 1, H₆),

7.08 (s, 1H, rotamer 2, H₆), 6.87 (t, 1H, rotamer 1, J= 5.8 Hz, H_12’),

6.81 (t, 1H, rotamer 2, J= 5.8 Hz, H_12’), 4.58 (s, 2H, rotamer 1, H_8’),

2-[(((N⁶-(benzoyl)adenin-9-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-(tert-butyloxycarbonyl-

amino)ethyl]acetamide (4c).

A mixture of 2-{[tert-butyl(diphenyl)silyl]oxy}ethanamine 1b (50 4.51 (s, 2H, rotamer 2, H_8’) 4.12 (s, 2H, rotamer 1, H_5’), 3.93 (s, 2H,

mg, 0.17 mmol, 1.0 equiv.) and paraformaldehyde (5.1 mg, 0.17 rotamer 2, H_5’), 3.83 (t, 2H, rotamer 1, J= 5.8 Hz, H_2’), 3.70 (t, 2H,

mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 334 μL) was rotamer 2, J= 5.8 Hz, H_2’), 3.57 (t, 2H, rotamer 1, J= 5.8 Hz, H_3’), 3.48

placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. (2- (t, 2H, rotamer 2, J= 5.8 Hz, H_3’), 3.13 (m, 2H, rotamer 1, H_10’), 3.06

Isocyano-ethyl)-carbamic acid tert-butyl ester 3a (28.8 mg, 0.17 (m, 2H, rotamer 2, H_10’), 3.06 (m, 2H, rotamer 1, H_11’), 2.98 (m, 2H,

mmol, 1.0 equiv.) and N⁶-(benzoyl)-9-(carboxymethyl)adenine 2c rotamer 2, H_11’) 1.75 (d, 3H, H₇), 1.39 (s, 9H, tBu-Boc), 1.02 (d, 9H,

(50.4 mg, 0.17 mmol, 1.0 equiv.) were then added dropwise. The tBu-TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 168.7 (C_7’, CO),

resulting solution was irradiated at 50 W (60 °C) for 3 h. The 168.1 (C_6’, CO), 165.2 (C₄, CO) 156.5 (C-Boc, CO) 151.8 (C₂, CO),

reaction mixture was concentrated to dryness under reduced 142.8 (C₆, CH), 135.9 (C_ortho-TBDPS, CH), 133.6 (C_ph-TBDPS, C), 130.8

pressure and the residue was purified by column chromatography (C_para-TBDPS, CH), 128.8 (C_meta-TBDPS, CH), 108.9 (C₅, C), 78.5 (C-

(DCM with 5% MeOH) to afford 4c as a white powder (52.9 mg, 41% tBu-Boc, C), 62.6 and 61.9 (C₂, CH₂, rotamers), 51.6 and 50.3 (C₅,

1

yield). Tr-HPLC: 24.1 min (>98%, 10-100% ACN in 40 min). H NMR CH₂, rotamers), 49.8 and 48.7 (C_3’CH₂, rotamers), 48.2 (C_8’, CH₂)

(400 MHz, DMSO-d₆): δ (ppm) 11.04 (bs, 1H, NH-Bz), 8.73 (s, 1H, H₂), 40.0 and 39.3 (C_11’, rotamers, CH₂), 39.5 and 39.3 (C_10’, CH₂,

8.32 (s, 1H, H₈), 8.26 (t, 1H, rotamer 1, J=5.4 Hz, H_9’), 7.94 (t, 1H, rotamers), 29.1 (C-tBu-Boc, CH₃), 27.5 (C-tBu-TBDPS, CH₃), 19.5 (C-

rotamer 2, J=6.0 Hz, H_9’), 8.08 (m, 2H, H_para-Bz), 7.67-7.64 (m, 1H, tBu-TBDPS, C), 12.7 (C₇, CH₃). HRMS ESI^-calcd for C₃₄H₄₆N₅O₇Si^-(M-

H_meta-Bz), 7.58-7.51 (m, 2H, H_ortho-Bz), 7.64-7.59 (m, 4H, H_ortho

-

H)^-664.3167, found 664.3166.

TBDPS), 7.46-7.42 (m, 6H, H_meta/para-TBDPS), 6.85 (t, 1H, rotamer 1,

J=5.4 Hz, H_12’), 6.72 (t, 1H, rotamer 2, J=5.4 Hz, H_12’), 5.40 (s, 2H, 2-[(((Uracil-1-yl)acetyl)-(2-(tert-

rotamer 1, H_8’), 5.23 (s, 2H, rotamer 2, H_8’), 4.17 (s, 2H, rotamer 1, butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-(tert-butyloxycarbonyl-

H_5’), 3.96 (s, 2H, rotamer 2, H_5’), 3.91 (t, 2H, rotamer 1, J= 5.4 Hz, amino)ethyl]acetamide (4e).

H_2’), 3.70 (t, 2H, rotamer 2, J= 6.1 Hz, H_2’), 3.62 (t, 2H, rotamer 1, J= A mixture of 2-{[tert-butyl(diphenyl)silyl]oxy}ethanamine 1b (100

5.4 Hz, H_3’), 3.48 (t, 2H, rotamer 2, J= 6.1 Hz, H_3’), 3.13 (m, 2H, mg, 0.33 mmol, 1.0 equiv.) and paraformaldehyde (10 mg, 0.33

rotamer 1, H_10’), 3.04 (m, 2H, rotamer 2, H_10’), 3.04 (m, 2H, mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 669 μL) was

rotamers 1, H_11’), 2.92 (m, 2H, rotamer 2, H_11’), 1.36 (s, 9H, tBu-Boc), placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. (2-

1.03 (s, 9H, tBu-TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) Isocyano-ethyl)-carbamic acid tert-butyl ester 3a (56.8 mg, 0.33

167.9 (C_6’, CO), 167.1 and 166.4 (C_7’, CO, rotamers), 166.3 (C-Bz, mmol, 1.0 equiv.) and uracil-1-acetic acid 2e (56.9 mg, 0.33 mmol,

CO), 155.6 (C-Boc, CO), 153.3 (C₄, C), 151.9 (C₂, C), 150.6 (C₆, C), 1.0 equiv.) were then added dropwise. The resulting solution was

146.1 (C₈, CH), 135.0 (C_ortho-TBDPS, CH), 134.2 (C_ph-Bz, C), 132.9 irradiated at 50 W (60 °C) for 3 h. The reaction mixture was

(C_meta-Bz, CH), 132.7 (C_ph-TBDPS), 129.8 (C_para-TBDPS, CH), 129.0 concentrated to dryness under reduced pressure and the residue

(C_ortho/para-Bz, CH), 128.0 (C_meta-TBDPS, CH), 125.0 (C₅, C), 77.6 (C- was purified by column chromatography (DCM with 5% MeOH) to

tBu-Boc, C), 61.2 and 60.9 (C_2’, CH₂, rotamers), 50.3 and 47.9 (C_5’, afford 4e as a white powder (159.4 mg, 73% yield). Tr-HPLC: 22.6

CH₂, rotamers), 49.3 and 49.2 (C_3’, CH₂, rotamers), 46.7 and 43.2 min (> 98%, 10-100% ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆):

(C_8’, CH_2,rotamers), 40.0 (C_11’, CH₂), 39.4 (C_10’, CH₂), 28.3 (C-tBu- δ (ppm) 11.27 (1H, s, H₃), 8.16 (t, 1H, rotamer 1, J= 5.8 Hz, H_9’), 7.84

Boc, CH₃), 26.6 (C-tBu-TBDPS, CH₃). HRMS ESI⁺calcd for (t, 1H, rotamer 2, J= 5.8 Hz, H_9’), 7.67 – 7.57 (m, 4H, H_ortho-TBDPS),

C₄₁H₅₁N₈O₆Si⁺(M+H)⁺779.3701, found 779.3708.

7.51 – 7.41 (m, 6H, H_meta/para-TBDPS), 7.40 (d, 1H, rotamer 1, H₆),

7.25 (d, 1H, rotamer 2, H₆), 6.83 (t, 1H, rotamer 1, J= 5.8 Hz, H_12’),

6.77 (t, 1H, rotamer 2, J= 5.8 Hz, H_12’), 5.55 (dd, 1H, H₅), 4.59 (s, 2H,

rotamer 1, H_8’), 4.53 (s, 2H, rotamer 2, H_8’), 4.10 (s, 2H, rotamer 1,

H_5’), 3.92 (s, 2H, rotamer 2, H_5’), 3.81 (t, 2H, rotamer 1, J= 5.8 Hz,

2-[(((Thymin-1-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-(tert-butyloxycarbonyl-

amino)ethyl]acetamide (4d).

A mixture of 2-{[tert-butyl(diphenyl)silyl]oxy}ethanamine 1b (150 H_2’), 3.69 (t, 2H, rotamer 2, J= 5.8 Hz, H_2’), 3.54 (t, 2H, rotamer 1, J=

mg, 0.50 mmol, 1.0 equiv.) and paraformaldehyde (15.1 mg, 0.50 5.8 Hz, H_3’), 3.47 (t, 2H, rotamer 2, J= 5.8 Hz, H_3’), 3.11 (m, 2H,

mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 1.0 mL) was rotamer 1, H_10’), 3.05 (m, 2H, rotamer 2, H_10’), 3.03 (m, 2H, rotamer

placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. (2- 1, H_11’), 2.97 (m, 2H, rotamer 2, H_11’), 1.37 (s, 9H, tBu-Boc), 0.99 (d,

Isocyano-ethyl)-carbamic Acid tert-butyl Ester 3a (85.3 mg, 0.50 9H, tBu-TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 168.2 (C_6’,

mmol, 1.0 equiv.) and thymin-1-acetic acid 2d (92.5 mg, 0.50 mmol, CO), 167.6 (C_7’, CO), 164.2 (C₄, CO), 156.1 (C-Boc, CO), 151.4 (C₂,

1.0 equiv.) were then added dropwise. The resulting solution was CO), 146.9 (C₆, CH), 135.5 (C_ortho-TBDPS, CH), 133.2 (C_ph-TBDPS, C),

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130.4 (C_para-TBDPS, CH), 128.4 (C_meta-TBDPS, CH), 101.1 (C₅, CH), °C) for 3 h. The reaction mixture was concentrated to dryness under

78.1 (CtBu-Boc, C), 62.6 and 61.2 (C_2’, CH₂, rotamer), 49.9 and 49.7 reduced pressure and the residue was purified by column

(C_3’, CH₂, rotamer), 49.4 (C_5’, CH₂), 48.4 (C_8’, CH₂), 39.7 (C_11’, CH₂), chromatography (DCM with 5% MeOH) to afford 4g as a brown

39.4 (C_10’, CH₂), 28.7 (C-tBu-Boc, CH₃), 27.1 (C-tBu-TBDPS, CH₃), 19.1 powder (123.5 mg, 62% yield). Tr-HPLC: 21.9 min (95%, 10-100%

(C-tBu-TBDPS, C). HRMS ESI^-calcd for C₃₃H₄₄N₅O₇Si^-(M-H)^-ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.28 (s, 1H,

650.3010, found 650.3015.

rotamer 1, H₃), 11.26 (s, 1H, rotamer 2, H₃), 8.62 (d, 1H, rotamer 1,

J= 5.8 Hz, H_9’), 8.32 (d, 1H, rotamer 2, J= 5.8 Hz, H_9’), 7.64-7.60 (m,

4H, H_ortho-TBDPS), 7.45-7.40 (m, 6H, H_meta/para-TBDPS), 7.22 (s, 1H,

2-[(((N⁴-(4-tert-butylbenzoyl)cytosin-1-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-(methylacetate)acetamide rotamer 1, H₆), 7.07 (s, 1H, rotamer 2, H₆), 4.56 (s, 2H, rotamer 1,

(4f).

H_8’), 4.51 (s, 2H, rotamer 2, H_8’), 4.21 (s, 2H, rotamer 1, H_5’), 4.02 (s,

A mixture of 2-{[tert-butyl(diphenyl)silyl

]

oxy

}ethanamine 1b (200 2H, rotamer 2, H_5’), 3.90 (d, 2H, rotamer 1, J= 5.8 Hz, H_10’), 3.85 (d,

mg, 0.67 mmol, 1.0 equiv.) and paraformaldehyde (20.1 mg, 0.67 2H, rotamer 2, J= 5.8 Hz, H_10’), 3.84 (t, 2H, rotamer 1, J= 5.4 Hz, H_2’),

mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 1.3 mL) was 3.71 (t, 2H, rotamer 2, J= 5.9 Hz, H_2’), 3.63 (s, 3H, OCH₃), 3.54 (t, 2H,

placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. rotamer 1, J= 5.4 Hz, H_3’), 3.48 (t, 2H, rotamer 2, J= 5.9 Hz, H_3’), 1.74

Methyl isocyanoacetate 3b (60.8 μL, 0.67 mmol, 10 equiv.) and N⁴- (d, 3H, rotamer 1, J= 1.0 Hz, H₇), 1.70 (d, 3H, rotamer 2, J= 1.0 Hz,

(4-tert-butylbenzoyl)-1-(carboxymethyl)cytosine 2b (220.2 mg, 0.67 H₇), 1.01-0.98 (s, 9H, tBu-TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ

mmol, 1.0 equiv.) were then added dropwise. The resulting solution (ppm) 170.2 (C_11’, CO), 168.7 (C_6’, CO), 167.7 (C_7’, CO), 164.4 (C₄,

was irradiated at 50 W (60 °C) for 3 h. The reaction mixture was CO), 151.0 (C₂, CO), 142.0 (C₆, CH), 135.1 (C_ortho-TBDPS, CH), 132.9

concentrated to dryness under reduced pressure and the residue (C_ph-TBDPS, C), 129.9 (C_para-TBDPS, CH), 127.9 (C_meta-TBDPS, CH),

was purified by column chromatography (DCM with 5% MeOH) to 108.1 (C₅, C), 61.7 and 61.1 (C_2’, CH₂, rotamers), 51.8 (OCH₃, CH₃),

afford 4f as a white powder (251.6 mg, 51% yield). Tr-HPLC: 25.9 50.5 and 48.6 (C_5’, CH₂, rotamers), 49.4 and 49.2 (C_3’, CH₂,

min (95 %, 10-100% ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆):δ rotamers), 47.7 (C_8’, CH2), 40.7 and 40.4 (C_10’, CH₂, rotamers), 26.7

(ppm) 11.12 (1H, s, NH-tBuBz), 8.67 (d, 1H, rotamer 1, J= 5.9 Hz, and 26.6 (C-tBu-TBDPS, CH₃), 18.7 (C-tBu-TBDPS, C), 11.9 (C₇, CH₃).

H_9’), 8.32 (d, 1H, rotamer 2, J= 5.9 Hz, H_9’), 7.98 (d, 1H, J= 8.5 Hz, HRMS ESI⁺calcd for C₃₀H₃₉N₄O₇Si⁺(M+H)⁺595.2588, found

H_ortho-tBuBz), 7.94 (d, 1H, rotamer 1, J= 7.4 Hz, H₆), 7.83 (d, 1H, 595.2593.

rotamer 2, J= 7.4 Hz, H₆), 7.64-7.59 (m, 4H, H_ortho-TBDPS), 7.54 (d,

1H, J= 8.5 Hz, H_meta-tBuBz), 7.46-7.42 (m, 6H, H_meta/para-TBDPS), 7.32 2-[(((Uracil-1-yl)acetyl)-(2-(tert-

(d, 1H, J= 7.4 Hz, H₅), 4.71 (s, 2H, H_8’), 4.29 (s, 2H, rotamer 1, H_5’), butyl(diphenylsilyl)oxy)ethyl))]amino-N-(methylacetate)acetamide

4.07 (s, 2H, rotamer 2, H_5’), 3.92 (d, 2H, rotamer 1, J= 5.9 Hz, H_10’), (4h).

3.86 (d, 2H, rotamer 2, J= 5.9 Hz, H_10’), 3.90 (t, 2H, rotamer 1, J= 5.9 A mixture of 2-{[tert-butyl(diphenyl)silyl]oxy}ethanamine 1b (100

Hz, H_2’), 3.73 (t, 2H, rotamer 2, J= 5.9 Hz, H_2’), 3.64 (s, 3H, OCH₃), mg, 0.33 mmol, 1.0 equiv.) and paraformaldehyde (10 mg, 0.33

3.60 (t, 2H, rotamer 1, J= 5.9 Hz, H_3’), 3.51 (t, 2H, rotamer 2, J= 5.9 mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 668 μL) was

Hz, H_3’), 1.3 (s, 9H, tBuBz), 1.03 (s, 9H, tBu-TBDPS). ¹³C NMR (100 placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h.

MHz, DMSO-d₆): δ (ppm) 170.1 (C_11’, CO), 168.6 (C_6’, CO), 167.3 (C_7’, Methyl isocyanoacetate 3b (30.4 μL, 0.33 mmol, 1.0 equiv.) and

CO), 167.0 (C-tBuBz, CO), 163.3 (C₄, C), 155.7 (C_para-tBuBz, C), 151.0 uracil-1-acetic acid 2e (56.9 mg, 0.33 mmol, 1.0 equiv.) were then

and 150.8 (C₆, CH, rotamers), 135.0 (C_ortho-TBDPS, CH), 132.7 (C_ph

-

added dropwise. The resulting solution was irradiated at 50 W (60

TBDPS, C), 130.4 (C_ph-tBuBz, C), 129.9 (C_para-TBDPS, CH), 128.3 °C) for 3 h. The reaction mixture was concentrated to dryness under

(C_ortho- tBuBz, CH), 128.0 (C_meta-TBDPS, CH), 125.2 (C_meta- tBuBz, CH), reduced pressure and the residue was purified by column

95.7 (C₅, CH), 61.9 and 61.1 (C_2’, CH₂, rotamers), 51.8 (OCH₃, CH₃), chromatography (DCM with 5% MeOH) to afford 4h as a brown

49.7 (C_8’, CH₂), 49.4 (C_3’, CH₂), 50.8 and 48.9 (C_5’, CH₂, rotamers), powder (105.5 mg, 54% yield). Tr-HPLC: 21.2 min (95%, 10-100%

40.7 and 40.4 (C_10’, CH₂, rotamers), 34.8 (C-tBu-tBuBz, C), 30.8 (C- ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.02 (s, 1H,

tBu-tBuBz, CH₃), 26.6 (C-tBu-TBDPS, CH₃), 18.7 (C-tBu-TBDPS, C). H₃), 8.64 (t, 1H, rotamer 1, J= 5.9 Hz, H_9’), 8.33 (t, 1H, rotamer 2, J=

HRMS ESI⁺calcd for C₄₀H₅₀N₅O₇Si⁺(M+H)⁺740.3480, found 5.9 Hz, H_9’), 7.63-7.59 (m, 4H, H_ortho-TBDPS), 7.50-7.38 (m, 6H,

740.3482.

H_meta/para-TBDPS), 7.39 (d, 1H, rotamer 1, J= 1.0 Hz, H₆), 7.27 (d, 1H,

rotamer 2, J= 1.0 Hz, H₆), 5.57 (d, 1H, rotamer 1, J= 1.0 Hz, H₅), 5.51

(d, 1H, rotamer 2, J= 1.0 Hz, H₅), 4.57 (s, 2H, rotamer 1, H_8’), 4.54 (s,

2-[(((Thymin-1-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-(methylacetate)acetamide 2H, rotamer 2, H_8’), 4.20 (s, 2H, rotamer 1, H_5’), 4.01 (s, 2H, rotamer

(4g).

2, H_5’), 3.89 (d, 2H, rotamer 1, J= 5.9 Hz, H_10’), 3.83 (d, 2H, rotamer

A mixture of 2-{[tert-butyl (diphenyl) silyl

]

oxy

}ethanamine 1b (100 2, J= 5.9 Hz, H_10’), 3.82 (t, 2H, rotamer 1, J= 5.8 Hz, H_2’), 3.69 (t, 2H,

mg, 0.33 mmol, 1.0 equiv.) and paraformaldehyde (10 mg, 0.33 rotamer 2, J= 5.8 Hz, H_2’), 3.62 (s, 3H, OCH₃), 3.52 (t, 2H, rotamer 1,

mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 668 μL) was J= 5.8 Hz, H_3’), 3.47 (t, 2H, rotamer 2, J= 5.8 Hz, H_3’), 1.00 (d, 9H,

placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. tBu-TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 171.0 (C_11’, CO),

Methyl isocyanoacetate 3b (30.4 μl, 0.33 mmol, 1.0 equiv.) and 169.4 (C_6’, CO), 168.5 (C_7’, CO), 164.6 (C₄, CO), 151.8 (C₂, CO), 147.1

thymin-1-acetic acid 2d (61.7 mg, 0.33 mmol, 1.0 equiv.) were then (C₆, CH), 135.9 (C_ortho-TBDPS, CH), 133.6 (C_ph-TBDPS, C), 130.8 (C_para

-

added dropwise. The resulting solution was irradiated at 50 W (60 TBDPS, CH), 128.8 (C_meta-TBDPS, CH), 101.5 (C₅, CH), 62.6 and 61.9

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Journal Name

(C_2’, CH₂, rotamers), 52.6 (OCH₃, CH₃), 51.4 and 50.2 (C_5’, CH₂, δ (ppm) 11.19 (s, 1H, NH-Bz), 8.72 (s, 1H, H₂), 8.77 (t, 1H, rotamer 1,

rotamers), 49.5 and 48.7 (C_3’, CH₂, rotamers), 48.7 and 48.6 (C_8’, H_9’), 8.42 (t, 1H, rotamer 2, H_9’), 8.33 (s, 1H, H₈), 8.08 (m, 2H, H_para

-

CH₂, rotamers) 41.6 and 41.3 (C_10’, CH₂, rotamers), 27.5 (C-tBu- Bz), 7.67-7.64 (m, 1H, H_meta-Bz), 7.58-7.51 (m, 2H, H_ortho-Bz), 6.84 (t,

TBDPS, CH₃), 19.5 (C-tBu-TBDPS, C). HRMS ESI⁺calcd for 1H, rotamer 1, H_1’), 6.75 (t, 1H, rotamer 2, H_1’), 5.37 (s, 2H, rotamer

C₂₉H₃₇N₄O₇Si⁺(M+H)⁺581.2432, found 581.2438.

1, H_8’), 5.22 (s, 2H, rotamer 2, H_8’), 4.29 (s, 2H, rotamer 1, H_5’), 4.02

(s, 2H, rotamer 2, H_5’), 3.98 (t, 2H, rotamer 1, J= 5.8 Hz, H_10’), 3.87 (t,

2H, rotamer 2, J= 5.8 Hz, H_10’), 3.64 (s, 3H, OCH₃), 3.54 (m, 2H,

rotamer 1, H_3’), 3.34 (m, 2H, rotamer 2, H_3’), 3.15 (m, 2H, rotamer 1,

H_2’), 3.07 (m, 2H, rotamer 2, H_2’), 1.38 (s, 9H, tBu-Boc). ¹³C NMR

2-[(((N⁴-(4-tert-butylbenzoyl)cytosin-1-yl)acetyl)-(2-(tert-

butyloxycarbonyl-amino)ethyl))]amino-N-

(methylacetate)acetamide (4i).

A mixture of tert-butyl N-(2-aminoethyl)carbamate 1a (50 mg, 0.31 (100 MHz, DMSO-d₆): δ (ppm) 170.7 (C_11’, CO), 169.1 (C₅, CO), 166.7

mmol, 1.0 equiv.) and paraformaldehyde (9.4 mg, 0.31 mmol, 1.0 (C_7’, CO), 166.3 (C-Bz, CO), 156.2 (C-Boc, CO), 153.3 (C₄, C), 151.9

equiv.) in anhydrous isopropanol (0,5 M, 625 μL) was placed in a (C₂, CH), 150.6 (C₆, C), 146.0 (C₈, CH), 134.2 (C_ph-Bz, C), 132.9 (C_meta

-

sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. Methyl Bz, CH), 129.0 (C_para-Bz, CH), 129.0 (C_ortho-Bz, CH), 125.7 (C₅, C), 77.8

isocyanoacetate 3b (28.4 μL, 0.31 mmol, 1.0 equiv.) and N⁴-(4-tert- (C-tBu-Boc, C), 52.2 (OCH₃, CH₃), 50.5 and 48.9 (C_5’, CH₂, rotamers),

butylbenzoyl)-1-(carboxymethyl)cytosine 2b (113.0 mg, 0.31 mmol, 47.7 (C_3’, CH2), 44.4 and 44.2 (C_8’, CH₂, rotamers), 41.3 and 40.9

1.0 equiv.) were then added dropwise. The resulting solution was (C_10’, CH₂, rotamers), 38.4 and 37.9 (C_2’, CH₂, rotamers), 28.8 (C-tBu-

-

irradiated at 50 W (60 °C) for 3 h. The reaction mixture was Boc, CH₃). HRMS ESI^-calcd for C₂₆H₃₁N₈O₇(M-H)^-567.2316, found

concentrated to dryness under reduced pressure and the residue 567.2316.

was purified by column chromatography (DCM with 5% MeOH) to

afford 4i as a brown powder (69.3 mg, 37% yield). Tr-HPLC: 20.2 2-[(((Thymin-1-yl)acetyl)-(2-(tert-butyloxycarbonyl-

min (> 95%, 10-100% ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆): amino)ethyl))]amino-N-(methylacetate)acetamide (4k).

δ (ppm) 11.1 (s, 1H, NH-tBuBz), 8.68 (d, 1H, rotamer 1, J= 5.8 Hz, A mixture of tert-butyl N-(2-aminoethyl)carbamate 1a (100 mg, 0.63

H_9’), 8.35 (d, 1H, rotamer 2, J= 5.8 Hz, H_9’), 7.98 (d, 2H, J= 8.8 Hz, mmol, 1.0 equiv.) and paraformaldehyde (18.7 mg, 0.63 mmol, 1.0

H_ortho-tBuBz), 7.95 (m, 1H, H₆), 7.53 (d, 2H, J= 8.8 Hz, H_meta-tBuBz), equiv.) in anhydrous isopropanol (0.5 M, 1.25 mL) was placed in a

7.33 (m, 1H, H₅), 6.96 (t, 1H, rotamer 1, J= 5.8 Hz, H_1’), 6.72 (t, 1H, sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. Methyl

rotamer 2, J= 5.8 Hz, H_1’), 4.85 (s, 2H, rotamer 1, H_8’), 4.68 (s, 2H, isocyanoacetate 3b (56.8 μL, 0.63 mmol, 1.0 equiv.) and thymin-1-

rotamer 2, H_8’), 4.19 (s, 2H, rotamer 1, H_5’), 4.00 (s, 2H, rotamer 2, acetic acid 2d (115.2 mg, 0.63 mmol, 1.0 equiv.) were then added

H_5’), 3.94 (d, 1H, rotamer 1, J= 5.8 Hz, H_10’), 3.86 (d, 1H, rotamer 2, dropwise. The resulting solution was irradiated at 50 W (60 °C) for 3

J= 5.8 Hz, H_10’), 3.65 (s, 3H, OCH₃), 3.43 (t, 2H, rotamer 1, J= 6.8 Hz, h. The reaction mixture was concentrated to dryness under reduced

H_3’), 3.34 (t, 2H, rotamer 2, J= 6.8 Hz, H_3’), 3.22 (dt, 2H, rotamer 1, J= pressure and the residue was purified by column chromatography

5.8-6.8 Hz, H_2’), 3.05 (dt, 2H, rotamer 2, J= 5.8-6.8 Hz, H_2’), 1.39 (s, (DCM with 5% MeOH) to afford 4k as a brown powder (128.9 mg,

9H, tBu-Boc), 1.32 (s, 9H, tBuBz). ¹³C NMR (100 MHz, DMSO-d₆): δ 45% yield). Tr-HPLC: 14.3 min (97%, 10-100% ACN in 40 min). ¹H

(ppm) 170.0 (C_11’, CO), 168.7 (C_6’, CO), 167.6 (C_7’, CO), 167.0 (C- NMR (400 MHz, DMSO-d₆): δ (ppm) 11.28 (s, 1H, H₃), 8.69 (t, 1H,

tBuBz, CO), 163.3 (C₄, C), 155.7 (C-Boc, CO), 155.7 (C_para-tBuBz, C), rotamer 1, H_9’), 8.40 (t, 1H, rotamer 2, H_9’), 7.32 (s, 1H, H₆), 7.00 (t,

155.2 (C₂, CO), 151.0 (C₆, CH), 130.3 (C_ph-tBuBz, C), 128.3 (C_ortho

-

1H, rotamer 1, H_1’), 6.77 (t, 1H, rotamer 2, H_1’), 4.66 (s, 2H, rotamer

tBuBz, CH), 125.2 (C_meta-tBuBz, CH), 95.7 (C₅, CH), 77.8 (C-tBu-Boc, 1, H_8’), 4.50 (s, 2H, rotamer 2, H_8’), 4.14 (s, 2H, rotamer 1, H_5’), 3.99

C), 50.2 and 48.6 (C_5’, CH₂, rotamers), 51.6 (OCH₃, CH3), 49.6 (C_8’, (s, 2H, rotamer 2, H_5’), 3.95 (d, 2H, rotamer 1, J= 5.8 Hz, H_10’), 3.88

CH₂), 47.2 (C_3’, CH₂), 40.8 and 40.5 (C_10’, CH₂, rotamers), 40.0 and (d, 2H, rotamer 2, J= 5.8 Hz, H_10’), 3.66 (s, 3H, OCH₃), 3.39 (m, 2H,

38.0 (C_2’, CH₂, rotamers), 34.8 (C-tBu-tBuBz, C), 30.8 (C-tBu-tBuBz, rotamer 1, H_3’), 3.34 (m, 2H, rotamer 2, H_3’), 3.19 (m, 2H, rotamer 1,

CH₃), 28.2 (C-tBu-Boc, CH₃). HRMS ESI⁺calcd for C₂₉H₄₁N₆O₈⁺(M+H)⁺H_2’), 3.08 (m, 2H, rotamer 2, H_2’), 1.77 (s, 3H, H₇), 1.40 (s, 9H, tBu-

601.2986, found 601.2987.

Boc). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 170.6 (C_11’, CO), 169.2

(C_8’, CO), 168.2 (C_7’, CO), 164.8 (C₄, CO), 156.1 (C-Boc, CO), 151.5

(C₂, CO), 142.5 (C₆, CH), 108.6 (C₅, C), 78.4 (C-tBu-Boc, C), 52.2

(OCH₃, CH₃), 50.4 and 48.8 (C_5’, CH₂, rotamers), 48.2 and 48.0 (C_8’,

2-[(((N⁶-(benzoyl)adenin-9-yl)acetyl)-(2-(tert-butyloxycarbonyl-

amino)ethyl))]amino-N-(methylacetate)acetamide (4j).

A mixture of tert-butyl N-(2-aminoethyl)carbamate 1a (100 mg, 0.63 CH₂, rotamers), 47.6 and 47.4 (C_3’, CH₂, rotamers), 41.2 and 40.9

mmol, 1.0 equiv.) and paraformaldehyde (18.7 mg, 0.63 mmol, 1.0 (C_10’, CH₂, rotamers), 38.3 and 37.9 (C_2’, CH₂, rotamers), 28.7 (C-tBu-

equiv.), in anhydrous isopropanol (0,5 M, 1.25 mL) was placed in a Boc, CH₃), 12.4 (C₇, CH₃). HRMS ESI^-calcd for C₁₉H₂₈N₅O₈(M-H)^-454.

sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. Methyl 1938, found 454. 1943.

isocyanoacetate 3b (56.8 μL, 0.63 mmol, 1.0 equiv.) and N⁶-

(benzoyl)-9-(carboxymethyl)adenine 2c (185.5 mg, 0.63 mmol, 1.0

equiv.) were then added dropwise. The resulting solution was 2-[(((Uracil-1-yl)acetyl)-(2-(tert-butyloxycarbonyl-

irradiated at 50 W (60 °C) for 3 h. The reaction mixture was amino)ethyl))]amino-N-(methylacetate)acetamide (4l).

concentrated to dryness under reduced pressure and the residue A mixture of tert-butyl N-(2-aminoethyl)carbamate 1a (100 mg, 0.63

was purified by column chromatography (DCM with 5% MeOH) to mmol, 1.0 equiv.) and paraformaldehyde (18.7 mg, 0.63 mmol, 1.0

afford 4j as a brown powder (141.9 mg, 40% yield). Tr-HPLC: 15.9 equiv.) in anhydrous isopropanol (0.5 M, 1.25 mL) was placed in a

min (> 88%, 10-100% ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆): sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. Methyl

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ARTICLE

isocyanoacetate 3b (56.8 μl, 0.63 mmol, 1.0 equiv.) and uracil-1- 52.2 (OCH₃, CH₃), 50.1 (C_8’, CH₂), 48.9 (C_5’, CH₂), 48.9 (C_3’, CH₂), 40.8

acetic acid 2e (106.2 mg, 0.63 mmol, 1.0 equiv.) were then added and 41.2 (C_10’, CH₂), 40.9 (C_2’, CH₂), 35.4 (C-tBuBz, C), 31.4 (C-tBuBz,

+

dropwise. The resulting solution was irradiated at 50 W (60 °C) for 3 CH₃). HRMS ESI⁺calcd for C₄₄H₄₉N₆O₇(M+H)⁺773.3663, found

h. The reaction mixture was concentrated to dryness under reduced 773.3654.

pressure and the residue was purified by column chromatography

(DCM with 5% MeOH) to afford 4l as a brown powder (111.3 mg, 2-[(((N²-(isobutanoyl)guanin-9-yl)acetyl)-(2-(tert-

1

41% yield). Tr-HPLC: 13.5 min (> 99%, 10-100% ACN in 40 min). H butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-[

(9H-fluoren-9-yl)-

NMR (400 MHz, DMSO-d₆): δ (ppm) 11.30 (s, 1H, H₃), 8.71 (t, 1H, methoxy)carbonyl]-amino)ethyl]acetamide (4n)

rotamer 1, H_9’), 8.42 (t, 1,H rotamer 2, H_9’), 7.46 (s, 1H, rotamer 1, A mixture of 2-{[tert-butyl(diphenyl)silyl

]

oxy

}

ethanamine 1b (45.8

H₆), 7.40 (s, 1H, rotamer 2, H₆), 6.99 (t, 1H, rotamer 1, H_1’), 6.77 (t, mg, 0.15 mmol, 1.0 equiv.) and paraformaldehyde (4.6 mg, 0.15

1H, rotamer 2, H_1’), 5.62-5.59 (m, 1H, H₅), 4.71 (s, 2H, rotamer 1, mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 500 μL) was

H_8’), 4.54 (s, 2H, rotamer 2, H_8’), 4.15 (s, 2H, rotamer 1, H_5’), 3.99 (s, placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. (2-

2H, rotamer 2, H_5’), 3.95 (d, 2H, rotamer 1, J= 5.8 Hz, H_10’), 3.88 (d, Isocyano-ethyl)-carbamic acid 9H-fluoren-9-yl-methyl ester 3c (44.7

2H, rotamer 2, J= 5.8 Hz, H_10’), 3.66 (s, 3H, OCH₃), 3.31 (m, 2H, mg, 0.15 mmol, 1.0 equiv.) and N²-(isobutanoyl)-9-(carboxymethyl)

rotamer 1, H_3’), 3.19 (m, 2H, rotamer 2, H_3’), 3.19 (m, 2H, rotamer 1, guanine 2a (42.7 mg, 0.15 mmol, 1.0 equiv.) were then added

H_2’), 3.01 (m, 2H, rotamer 2, H_2’), 1.40 (s, 9H, tBu-Boc). ¹³C NMR dropwise. The resulting solution was irradiated at 50 W (60 °C) for 3

(100 MHz, DMSO-d₆): δ (ppm) 170.1 (C_11’, CO), 168.7 (C_6’, CO), 167.6 h. The reaction mixture was concentrated to dryness under reduced

(C_7’, CO), 163.8 (C₄, CO), 155.7 (C-Boc, CO), 151.0 (C₂, CO), 146.3 (C₆, pressure and the residue was purified by column chromatography

CH), 100.6 (C₅, CH), 78.0 (C-tBu-Boc, C), 51.7 (OCH₃, CH₃), 50.1 and (DCM with 5% MeOH) to afford 4l as a white powder (57.4 mg, 43%

48.3 (C_5’, CH₂, rotamers), 47.9 and 47.8 (C_8’, CH₂, rotamers), 47.7 yield). HPLC/MS ESI⁺/ESI^-883.6 [M+H] ⁺; 882.46 [M-H]^-.

and 47.1 (C_3’, CH₂, rotamers), 40.7 and 40.4 (C_10’, CH₂, rotamers),

37.8 and 37.5 (C_2’, CH₂, rotamers), 28.2 (C-tBu-Boc, CH₃). HRMS ESI^-General procedure for deprotection of PNA monomers 5

calcd for C₁₈H₂₆N₅O₈^-(M-H)^-440.1781, found 440.1787.

PNA monomer 4 was dissolved in a 50% TFA/DCM solution and the

2-[(((N⁴-(4-tert-butylbenzoyl)cytosin-1-yl)acetyl)-(2-(4-

monomethoxytrityl)oxy)ethyl))]amino-N-

(methylacetate)acetamide (4m).

mixture was stirred 1 h at 0 °C. After evaporation of the TFA, the

residue was taken up in DCM and washed with saturated NaHCO₃

solution. The organic layer was dried over Na₂SO₄, filtered and

A mixture of 2-{[4-monomethoxytrityl]oxy}ethanamine 1d (56.1 evaporated. The residue was then taken up in DCM and scavenger

mg, 0.17 mmol, 1.0 equiv.) and paraformaldehyde (5.1 mg, 0.17 resin benzyltriammonium chloride (3 equiv./mmol of 4) was added.

mmol, 1.0 equiv.) in anhydrous isopropanol (0,5 M, 600 μl) was The mixture was stirred 3 h at rt. The resin was filtered, washed

placed in a sealed vessel (0.5 – 2 mL) and stirred at rt for 1 h. with DCM and the filtrate was evaporated to give the deprotected

Methyl isocyanoacetate 3b (15.3 μl, 0.17 mmol, 1.0 equiv.) and N⁴- PNA monomer 5 as a white powder.

(4-tert-butylbenzoyl)-1-(carboxymethyl)cytosine 2b (55.6 mg, 0.17

mmol, 1.0 equiv.) were then added dropwise. The resulting solution 2-[(((N²-(isobutanoyl)guanin-9-yl)acetyl)-(2-(tert-

was irradiated at 50 W (60 °C) for 3 h. The reaction mixture was butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-aminoethyl]acetamide

concentrated to dryness under reduced pressure and the residue (5a)

was purified by column chromatography (DCM with 5% MeOH) to Following the general procedure, compound 4a (550 mg, 0,72

afford 4m as a brown powder (53.3 mg, 41% yield). Tr-HPLC: 24.9 mmol) afforded 5a as a white powder (400 mg, 84% yield). Tr-

min (99%, 10-100% ACN in 40 min). ¹H NMR (400 MHz, DMSO-d₆): δ HPLC/MS: 7.7 min (> 99%, 2-100% ACN in 9 min). ¹H NMR (400

(ppm) 11.07 (s, 1H, NH-tBuBz), 8.65 (d, 1H, rotamer 1, J= 5.8 Hz, MHz, DMSO-d₆) δ (ppm) 12.06 (bs, 1H, H₁), 11.66 (bs, 1H, NH-iBu),

H_9’), 8.26 (d, 1H, rotamer 2, J= 5.8 Hz, H_9’), 7.94 (s, 1H, H₆), 7.93 (d, 8.28 (t, 1H, rotamer 1, J=5.3 Hz, H_9’), 7.91 (t, 1H, rotamer 2, J=5.3

2H, J= 8.8 Hz, H_ortho-tBuBz), 7.50 (d, 2H, J= 8.8 Hz, H_meta-tBuBz), 7.33 Hz, H_9’), 7.76 (s, 1H, rotamer 1, H₈), 7.46 (s, 1H, rotamer 2, H₈), 7.64-

(s, 1H, H₅), 7.42-76.81 (m, 14H, H_arom-Mmt), 5.71 (s, 1H, H_1’), 4.98 (s, 7.60 (m, 4H, H_ortho-TBDPS), 7.47-7.42 (m, 6H, H_meta/para-TBDPS), 5.04

2H, rotamer 1, H_8’), 4.66 (s, 2H, rotamer 2, H_8’), 4.31 (s, 2H, rotamer (s, 2H, rotamer 1, H_8’), 4.96 (s, 2H, rotamer 2, H_8’), 4.20 (s, 2H,

1, H_5’), 3.91 (s, 2H, rotamer 2, H_5’), 3.87 (d, 2H, rotamer 1, J= 5.9 Hz, rotamer 1, H_5’), 3.93 (s, 2H, rotamer 2, H_5’), 3.89 (t, 2H, rotamer 1,

H_10’), 3.90 (t, 2H, rotamer 2, J= 5.9 Hz, H_10’), 3.73 (t, 2H, rotamer 2, J= 5.4 Hz, H_2’), 3.70 (t, 2H, rotamer 2, J= 6.1 Hz, H_2’), 3.77 (bs, 2H,

J= 5.9 Hz, H_2’), 3.69-3.67 (s, 3H, OCH₃), 3.62-3.60 (s, 3H, OCH₃), 3.48 H_12’), 3.64 (t, 2H, rotamer 1, J= 5.4 Hz, H_3’), 3.50 (t, 2H, rotamer 2, J=

(m, 2H, rotamer 1, H_3’), 3.29 (m, 2H, rotamer 2, H_3’), 2.11 (m, 2H, 6.1 Hz, H_3’), 3.17 (m, 2H, rotamer 1, H_10’), 3.07 (m, 2H, rotamer 2,

H_2’), 1.27 (s, 9H, tBuBz). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) H_10’), 2.68 (m, 2H, rotamer 1, H_11’), 2.55 (m, 2H, rotamer 2, H_11’),

170.7 (C_11’, CO), 169.1 (C_6’, CO), 167.9 (C_7’, CO), 167.7 (C-tBuBz, CO), 2.71 (sept, 1H, J= 6.9 Hz, H-iBu), 1.10 (d, 6H, J=6.9 Hz, iBu), 1.03 (s,

157.9 (C₂, CO), 156.3 (C_h-Mmt, C), 151.6 (C_para-tBuBz, C), 146.91 (C_a- 9H, tBu-TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 180.6 (C-

Mmt, C), 138.4 (C_e-Mmt, C), 130.9 (C_ph-tBuBz, C), 130.2 (C_ortho

-

iBu, CO), 168.0 (C_6’, CO), 167.1 and 166.5 (C_7’, CO, rotamers), 155.2

tBuBz, CH), 130.05 (C_f-Mmt, CH), 128.9 (C_c-Mmt, CH), 128.2 (C_b- (C₆, CO), 149.5 (C₄, C), 148.7 (C₂, C), 140.4 and 139.8 (C₈, CH,

Mmt, CH), 126.5 (C_d-Mmt, CH), 125.8 (C_meta-tBuBz, CH), 113.5 (C_g- rotamers), 135.0 (C_ortho-TBDPS, CH), 132.7 (C_ph-TBDPS, C), 130.0

Mmt, CH), 96.2 (C₅, CH), 70.4 (C-Mmt, C), 55.4 (OCH₃-Mmt, CH₃), (C_para-TBDPS, CH), 128.0 (C_meta-TBDPS, CH), 119.5 (C₅, C), 61.9 and

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60.9 (C_2’, CH₂, rotamers), 50.7 and 49.1 (C_5’, CH₂, rotamers), 49.5 HPLC/MS: 7.5 min (> 95%, 2-100% ACN in 9 min). ¹H NMR (400

and 49.2 (C_3’, CH₂, rotamers), 44.4 and 44.0 (C_8’, CH₂, rotamers), MHz, DMSO-d₆) δ (ppm) 11.26 (1H, s, H₃), 8.17 (t, 1H, rotamer 1, J=

42.1 and 41.7 (C_10’, CH₂, rotamers), 41.0 and 40.8 (C_11’, CH₂, 5.8 Hz, H_9’), 7.88 (t, 1H, rotamer 2, J= 5.8 Hz, H_9’), 7.67 – 7.57 (m,

rotamers), 35.1 (C-iBu, CH), 26.7 (C-tBu-TBDPS, 3CH₃), 19.0 (C-iBu, 4H, H_ortho-TBDPS), 7.51 – 7.41 (m, 6H, H_meta/para-TBDPS), 7.40 (m, 1H,

2CH₃), 18.7 (C-tBu-TBDPS, C). HRMS ESI⁺calcd for C₃₃H₄₅N₈O₅Si⁺rotamer 1, H₆), 7.25 (m, 1H, rotamer 2, H₆), 5.52 (dd, 1H, H₅), 4.58

(M+H)⁺661.3282, found 661.3280. HPLC/MS ESI⁺/ESI^-661.10 [M+H] (s, 2H, rotamer 1, H_8’), 4.52 (s, 2H, rotamer 2, H_8’), 4.12 (s, 2H,

⁺; 659.46 [M-H]^-.

rotamer 1, H_5’), 3.90 (s, 2H, rotamer 2, H_5’), 3.77 (t, 2H, rotamer 1,

J= 5.8 Hz, H_2’), 3.66 (t, 2H, rotamer 2, J= 5.8 Hz, H_2’), 3.53 (t, 2H,

rotamer 1, J= 5.8 Hz, H_3’), 3.49 (t, 2H, rotamer 2, J= 5.8 Hz, H_3’), 3.25

(m, 2H, rotamer 1, H_10’), 3.04 (m, 2H, rotamer 2, H_10’), 3.03 (m, 2H,

rotamer 1, H_11’), 2.97 (m, 2H, rotamer 2, H_11’), 0.99 (d, 9H, tBu-

2-[(((N⁶-(benzoyl)adenin-9-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-aminoethyl]acetamide

(5c).

Following the general procedure, compound 4c (300 mg, 0.39 TBDPS). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 168.2 (C_6’, CO),

mmol) afforded 5c as a white powder (213 mg, 81% yield). Tr- 167.6 (C_7’, CO), 164.2 (C₄, CO), 151.4 (C₂, CO), 146.9 (C₆, CH), 135.5

HPLC/MS: 7.8 min (> 98%, 2-100% ACN in 9 min). ¹³C NMR (100 (C_ortho-TBDPS, CH), 133.3 (C_ph-TBDPS, C), 130.4 (C_para-TBDPS, CH),

MHz, DMSO-d₆) δ (ppm) 167.9 (C_6’, CO), 167.1 and 166.4 (C_7’, CO, 128.4 (C_meta-TBDPS, CH), 101.1 (C₅, CH), 61.2 (C_2’, CH₂), 49.9 (C_3’,

rotamers), 166.3 (C-Bz, CO), 153.3 (C₄, C), 151.9 (C₂, C), 150.6 (C₆, CH₂), 49.8 (C_5’, CH₂), 48.5 (C_8’, CH₂), 39.7 (C_11’, CH₂), 39.4 (C_10’, CH₂),

C), 146,1 (C₈, CH), 135.0 (C_ortho-TBDPS, CH), 133.4 (C_ph-Bz, C), 130.5 27.2 (C-tBu-TBDPS, CH₃), 19.2 (C-tBu-TBDPS, C). HRMS ESI⁺calcd for

(C_meta-Bz, CH), 130.4 (C_ph-TBDPS), 129.7 (C_para-TBDPS, CH), 129.0 C₂₈H₃₈N₅O₅Si⁺(M+H)⁺552.2637, found 552.2637. HPLC/MS ESI⁺/ESI^-

(C_ortho/para-Bz, CH), 128.0 (C_meta-TBDPS, CH), 125.0 (C₅, C), 61.2 and 551.99 [M+H] ⁺; 549.92 [M-H]^-.

60.9 (C_2’, CH₂, rotamers), 50.3 and 47.9 (C_5’, CH₂, rotamers), 49.3

and 49.2 (C_3’, CH₂, rotamers), 46.7 and 43.2 (C_8’, CH_2,rotamers),

40.0 (C_11’, CH₂), 39.4 (C_10’, CH₂), 26.6 (C-tBu-TBDPS, CH₃). HRMS ESI⁺Preparation of compounds 6a-f.

calcd for C₃₆H₄₃N₈O₄Si⁺(M+H)⁺679.3173, found 679.3171. HPLC/MS

ESI⁺/ESI^-678.81 [M+H] ⁺; 676.97 [M-H]^-.

2-[(((N²-(isobutanoyl)guanin-9-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-

2-[(((Thymin-1-yl)acetyl)-(2-(tert-

N-[2-[(((N⁴-(4-tert-butylbenzoyl)cytosin-1-yl)acetyl)-ethyl))]amino-

N-[(methylacetate)glycinamidyl ]acetamide (6a)

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-aminoethyl]acetamide

(5d).

A mixture of compound 5a (50 mg, 0.08 mmol, 1.0 equiv.) and

Following the general procedure, compound 4d (689 mg, 1.03 paraformaldehyde (2.5 mg, 0.08 mmol, 1.0 equiv.) in anhydrous

mmol) afforded 5d as a white powder (498.9 mg, 85% yield). Tr- isopropanol (0.5 M, 550 μL) was placed in a sealed vessel (0.5 – 2

HPLC/MS: 7.7 min (> 95%, 2-100% ACN in 9 min). ¹H NMR (400 mL) and stirred at rt for 1 h. Methyl isocyanoacetate (6.8 μL, 0.08

MHz, DMSO-d₆): δ (ppm) 11.27 (bs, 1H, H₃), 8.11 (t, 1H, rotamer 1, mmol,

1.0

equiv.)

and

N⁴-(4-tert-butylbenzoyl)-1-

J= 5.8 Hz, H_9’), 7.87 (t, 1H, rotamer 2, J= 5.8 Hz, H_9’), 7.65-7.57 (m, (carboxymethyl)cytosine 2b (25 mg, 0.08 mmol, 1.0 equiv.) were

4H, H_ortho-TBDPS), 7.53 – 7.42 (m, 6H, H_meta/para-TBDPS), 7.22 (s, 1H, then added dropwise. The resulting solution was irradiated at 100

rotamer 1, H₆), 7.10 (s, 1H, rotamer 2, H₆), 4.56 (s, 2H, rotamer 1, W (100 °C) for 45 min. The reaction mixture was concentrated to

H_8’), 4.47 (s, 2H, rotamer 2, H_8’) 4.12 (s, 2H, rotamer 1, H_5’), 4.06 (s, dryness under reduced pressure and the residue was purified by

2H, rotamer 2, H_5’), 3.88 (m, 2H, rotamer 1, H_2’), 3.78 (m, 2H, column chromatography (DCM with 10% MeOH) to afford 6a as a

rotamer 2, H_2’), 3.57 (t, 2H, rotamer 1, J= 5.8 Hz, H_3’), 3.48 (t, 2H, brown solid (36.3 mg, 43% yield). Tr-HPLC: 23.8 min (95%, 10-100%

rotamer 2, J= 5.8 Hz, H_3’), 3.25 (m, 2H, rotamer 1, H_10’), 3.06 (m, 2H, ACN in 40 min). ¹H NMR major conformer (600 MHz, 298 K, CD₃OD)

rotamer 2, H_10’), 3.06 (m, 2H, rotamer 1, H_11’), 2.99 (m, 2H, rotamer

(ppm): 7.94 (d, 2H, J=8.3 Hz, H_ortho-tBuBz), 7.77 (d, 1H, J=7.0 Hz, H₆

2, H_11’) 1.75 (d, 3H, H₇), 0.96 (m, 9H, tBu-TBDPS). ¹³C NMR (100 of C), 7.70 (d, 4H, J=7.1, H_ortho-TBDPS), 7.58 (2H, d, J=8.3 Hz, H_meta

-

MHz, DMSO-d₆): δ (ppm) 168.7 (C_7’, CO), 168.1 (C_6’, CO), 164.9 (C₄, tBuBz), 7.38-7.45 (m, 2H, H_para-TBDPS & m, 4H, H_meta-TBDPS), 7.30

CO), 151.5 (C₂, CO), 142.4 (C₆, CH), 135.6 (C_ortho-TBDPS, CH), 133.3 (s, 1H, H₈of G), 7.16 (d, 1H, J=7.0 Hz, H₅of C), 5.08 (s, 2H, H_8'of G),

(C_ph-TBDPS, C), 130.5 (C_para-TBDPS, CH), 128.5 (C_meta-TBDPS, CH), 4.73 (s, 2H, H_8'of C), 4.14 (bs, 2H, H_5'of C), 4.12 (bs, 2H, H_5'of G),

108.5 (C₅, C), 62.3 and 61.6 (C_2’, CH₂, rotamers), 51.3 and 49.9 (C_5’, 4.02 (t, 2H, H_2'of G), 3.96 (s, 2H, H_10'), 3.72 (2H, H_3'of G), 3.63 (s,

CH₂, rotamers), 49.8 and 48.9 (C_3’, CH₂, rotamers), 48.4 (C8’, CH₂) 3H, H_12'& m, 2H, H_3’of C), 3.51 (m, 2H, H_2'of C), 2.59 (m, 1H, CH-

40.0 and 39.3 (C_11’, CH₂, rotamers), 39.5 and 39.3 (C_10’, CH₂, iPrCO), 1.37 (s, 9H, CH₃-tBuBz), 1.18 (d, 6H, J=6.8 Hz, CH₃-iPrCO),

rotamers), 27.2 (C-tBu-TBDPS, CH₃), 19.2 (C-tBu-TBDPS, C), 12.5 (C₇, 1.11 (s, 9H, CH₃-TBDPS).

CH₃). HRMS ESI+ calcd for C₂₉H₄₀N₅O₅Si⁺(M+H)⁺566.2791, found

566.2793. HPLC/MS ESI⁺/ESI^-565.99 [M+H] ⁺; 564.01 [M-H]^-.

2-[(((N²-(isobutanoyl)guanin-9-yl)acetyl)-(2-(tert-

2-[(((Uracil-1-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-[((( Uracil-1-yl)acetyl)-

ethyl))]amino-N-[(methylacetate)glycinamidyl]acetamide (6b)

A mixture of compound 5a (411.9 mg, 0.62 mmol, 1.0 equiv.) and

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-aminoethyl]acetamide

(5e).

Following the general procedure, compound 4e (159 mg, 0.24 paraformaldehyde (20.6 mg, 0.62 mmol, 1.1 equiv.) in anhydrous

mmol) afforded 5e as a white powder (92.1 mg, 69% yield). Tr- isopropanol (0,5 M, 1.2 mL) was placed in a sealed vessel (0.5 – 2

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mL) and stirred at rt for 1 h. Methyl isocyanoacetate (56.7 μL, 0.62 (isobutanoyl)guanin-9-yl)acetyl)-ethyl))]amino-N-

mmol, 1.0 equiv.) and uracil-1-acetic acid 2e (106.2 mg, 0.62 mmol, [(methylacetate)glycinamidyl]acetamide (6d)

1.0 equiv.) were then added dropwise. The resulting solution was A mixture of compound 5c (100 mg, 0.15 mmol, 1.0 equiv.) and

irradiated at 100 W (100 °C) for 45 min. The reaction mixture was paraformaldehyde (4.9 mg, 0.15 mmol, 1.0 equiv.) in anhydrous

concentrated to dryness under reduced pressure and the residue isopropanol (0.5 M, 294 μL) was placed in a sealed vessel (0.5 – 2

was purified by column chromatography (DCM with 10% MeOH) to mL) and stirred at rt for 1 h. Methyl isocyanoacetate (13.4 μL, 0.15

afford 6b as a white solid (205,5 mg, 35% yield). Tr-HPLC: 20.5 min mmol, 1.0 equiv.) and N²-(isobutanoyl)-9-(carboxymethyl) guanine

(90%, 10-100% ACN in 40 min). HRMS ESI⁺calcd for C₄₄H₅₆N₁₁O₁₁Si⁺2a (41 mg, 0.15 mmol, 1.0 equiv.) were then added dropwise. The

(M+H)⁺942.3925, found 942.3925.

resulting solution was irradiated at 100 W (100 °C) for 45 min. The

reaction mixture was concentrated to dryness under reduced

pressure and the residue was purified by column chromatography

(DCM with 10% MeOH) to afford 6d as a brown solid (39.6 mg, 25%

yield). Tr-HPLC/MS: 8.8 min (< 80%, 2-100% ACN in 9 min). HRMS

ESI⁺calcd for C₅₂H₆₁N₁₄O₁₀Si⁺(M+H)⁺1069.4460, found 1069.4459.

2-[(((N⁶-(benzoyl)adenin-9-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-[(((N⁴-(4-tert-

butylbenzoyl)cytosin-1-yl)acetyl)-ethyl))]amino-N-

[(methylacetate)glycinamidyl]acetamide (6c)

A mixture of compound 5c (507.6 mg, 0.75 mmol, 1.0 equiv.) and

paraformaldehyde (22.4 mg, 0.75 mmol, 1.0 equiv.) in anhydrous 2-[(((Thymin-1-yl)acetyl)-(2-(tert-

isopropanol (0.5 M, 1.4 mL) was placed in a sealed vessel (0.5 – 2 butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-[(((Thymin-1-yl)acetyl)-

mL) and stirred at rt for 1 h. Methyl isocyanoacetate (68 μL, 0.75 ethyl))]amino-N-[(methylacetate)glycinamidyl]acetamide (6e)

mmol,

1.0

equiv.)

and

N⁴-(4-tert-butylbenzoyl)-1- A mixture of compound 5d (498.9 mg, 0.88 mmol, 1.0 equiv.) and

(carboxymethyl)cytosine 2b (246 mg, 0.75 mmol, 1.0 equiv.) were paraformaldehyde (26.5 mg, 0.88 mmol, 1.0 equiv.) in anhydrous

then added dropwise. The resulting solution was irradiated at 100 isopropanol (0.5 M, 1.7 mL) was placed in a sealed vessel (0.5 – 2

W (100 °C) for 45 min. The reaction mixture was concentrated to mL) and stirred at rt for 1 h. Methyl isocyanoacetate (80 μL, 0.88

dryness under reduced pressure and the residue was purified by mmol, 1.0 equiv.) and thymin-1-acetic acid 2d (163.2 mg, 0.88

column chromatography (DCM with 10% MeOH) to afford 6c as a mmol, 1.0 equiv.) were then added dropwise. The resulting solution

white solid (250.8 mg, 30% yield). Tr-HPLC: 24.7 min (98%, 10-100% was irradiated at 100 W (100 °C) for 45 min. The reaction mixture

1

ACN in 40 min). H NMR (400 MHz, 298 K, 15 mM, CDCl₃) (ppm): was concentrated to dryness under reduced pressure and the

9.43 (bs, 1H, H_N6of A), 8.69 (s, 1H, H₂of A), 8.69 (bs, 1H, H_N4of C), residue was purified by column chromatography (DCM with 10%

8.33 (bs, 1H, H_N1'of C), 8.23 (bs, 1H, H_N9'), 7.95 (d, 2H, J=7.6 Hz, MeOH) to afford 6e as a white solid (303.2 mg, 40% yield). Tr-HPLC:

H_ortho-Bz), 7.92 (s, 1H, H₈of A), 7.69 (dd, 4H, J=7.6 and 2.0 Hz, H_ortho

-

20.2 min (96%, 10-100% ACN in 40 min). HRMS ESI⁺calcd for

TBDPS), 7.62 (d, 2H, J=7.2 Hz, H_ortho-tBuBz), 7.55 (d, 1H, J=7.0 Hz, H₆C₄₁H₅₃N₈O₁₁Si⁺(M+H)⁺861.3599, found 861.3598.

of C), 7.42 (m, 2H, H_para-TBDPS), 7.39 (m, 4H, H_meta-TBDPS), 7.38 (2H,

H_meta-tBuBz), 7.37 (m, 1H, H_para-Bz), 7.20 (t, 2H, J=7.6 Hz, H_meta-Bz), 2-[(((Uracil-1-yl)acetyl)-(2-(tert-

7.00 (bs, 1H, H₅of C), 5.21 (s, 2H, H_8'of A), 4.59 (s, 2H, H_8'of C), 4.05 butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-[(((N⁴-(4-tert-

(d, 2H, J=5.6 Hz, H_10'), 4.05 (bs, 2H, H_5'of A), 4.01 (bs, 2H, H_5'of C), butylbenzoyl)cytosin-1-yl)acetyl)-ethyl))]amino-N-

3.94 (t, 2H, J=5.3 Hz, H_2'of A), 3.63 (bs, 2H, H_3'of A), 3.54 (s, 3H, [(methylacetate)glycinamidyl]acetamide (6f)

H_12'), 3.48 (bs, 2H, H_3'of C), 3.42 (bs, 2H, H_2'of C), 1.34 (s, 9H, tBu- A mixture of compound 5e (80 mg, 0.15 mmol, 1.0 equiv.) and

tBuBz), 1.11 (s, 9H, tBu-TBDPS). ¹³C{¹H} NMR (100 MHz, 298 K, 15 paraformaldehyde (4.3 mg, 0.15 mmol, 1.0 equiv.) in anhydrous

mM, CDCl₃) (ppm): 171.6 (C_6'of C, CO), 170.8 (C_11', CO), 168.0; isopropanol (0.5 M, 289 μL) was placed in a sealed vessel (0.5 – 2

167.9 & 167.8 (C_6'of A, CO; C_7'of C, CO & C_7'of A, CO), 166.4 (C- mL) and stirred at rt for 1 h. Methyl isocyanoacetate (13.2 μL, 0.15

tBuBz, CO), 165.2 (C-Bz, CO), 162.8 (C₄of C, C), 157.0 (C-tBuBz, C), mmol,

1.0

equiv.)

and

N⁴-(4-tert-butylbenzoyl)-1-

156.5 (C₂of C, C), 152.7 (C₂of A, CH), 152.5 (C4 of A, C), 151.4 (C₆of (carboxymethyl)cytosine 2b (47.8 mg, 0.15 mmol, 1.0 equiv.) were

C, CH), 149.5 (C₆of A, C), 145.4 (C₈of A, CH), 135.7 (C_ortho-TBDPS, then added dropwise. The resulting solution was irradiated at 100

CH), 133.8 (C-Bz, C), 132.7 (C-TBDPS, C), 132.3 (C_para-Bz, CH), 130.3 W (100 °C) for 45 min. The reaction mixture was concentrated to

(C_para-TBDPS, CH), 130.0 (C-tBuBz, C), 128.6 (C_meta-Bz, CH), 128.2 dryness under reduced pressure and the residue was purified by

(C_ortho-Bz, CH & C_meta-TBDPS, CH), 127.7 (C_ortho-tBuBz, CH), 125.9 column chromatography (DCM with 10% MeOH) to afford 6f as a

(C_meta-tBuBz, CH), 123.0 (C₅of A, C), 96.5 (C₅of C, CH), 61.6 (C_2'of A, white solid (34.4 mg, 25% yield). Tr-HPLC: 23.6 min (95%, 10-100%

CH₂), 52.4 (C_12', CH₃), 50.7 & 50.0 (C_5'of A, CH₂& C_5'of C, CH₂), 50.5 ACN in 40 min). HRMS ESI⁺calcd for C₅₀H₆₂N₉O₁₁Si⁺(M+H)⁺

(C_8'of C, CH₂& C_3'of A, CH₂), 48.9 (C_3'of C, CH₂), 44.4 (C_8'of A, CH₂), 992.4334, found 992.4333.

41.6 (C_10', CH₂), 36.3 (C_2'of C, CH₂), 35.3 (C-tBu-tBuBz_,C), 31.2 (C-

tBu-tBuBz, CH₃), 27.1 (CtBu-TBDPS, CH₃), 19.3 (CtBu-TBDPS, C). Preparation of compounds 7a-c and 7e.

HRMS ESI⁺calcd for C₅₈H₆₇N₁₂O₁₀Si⁺(M+H)⁺1119.4868, found

1119.4867.

2-[((Guanin-9-yl)acetyl)-(2-hydroxyethyl)]amino-N-[2-[((Cytosin-1-

yl)acetyl)-ethyl))]amino-N-[ (carbamoyl)glycinamidyl]acetamide

(7a)

2-[(((N⁶-(benzoyl)adenin-9-yl)acetyl)-(2-(tert-

butyl(diphenylsilyl)oxy)ethyl))]amino-N-[2-[(((N²-

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PNA dimer 6a (60 mg) was dissolved in THF/MeOH 1:3 (v:v) (400 μL) 2-[((Adenin-9-yl)acetyl)-(2-hydroxyethyl)]amino-N-[2-[((cytosin-1-

and 28% aqueous ammonia was added (600 μL). The flask was yl)acetyl)-ethyl))]amino-N-[(carbamoyl)glycinamidyl]acetamide

sealed and the mixture was stirred 6 h at 50 °C. The solvents were (7c)

then removed under reduced pressure and the crude material was PNA dimer 6c (50 mg) was dissolved in THF/MeOH 1:3 (v:v) (5 mL)

taken up in THF (500 μL). A solution of TBAF (1 M) in THF was added and 28% aqueous ammonia was added (3 mL). The flask was sealed

and the reaction mixture was stirred 1 h at room temperature. The and the mixture was stirred 6 h at 50 °C. The solvents were then

reaction mixture was concentrated to dryness under reduced removed under reduced pressure and the crude product was taken

pressure and the residue was purified by reverse phase column up in THF (3 mL). A solution of TBAF (1 M) in THF (440 μL) was

chromatography (0 to 100% of 0.05 M TEAB buffer (pH 7.5) in 50% added and the reaction mixture was stirred

1 h at room

ACN) to give PNA dimer 7a (12 mg, 33%). Tr-HPLC: 16.1 min (98%, 0- temperature. The reaction mixture was concentrated to dryness

100% TEAB (0.05 M) ACN/H₂O 50/50 in 55 min). ¹H NMR major under reduced pressure and the residue was purified by reverse

conformer (400 MHz, 323K, 15 mM, D₂O) (ppm): 7.69 (s, 1H, H₈of phase column chromatography (0 to 100% of 0.05 M TEAB buffer

G), 7.15 (d, 1H, J=7.3 Hz, H₆of C), 5.68 (d, 1H, J=7.3 Hz, H₅of C), (pH 7.5) in 50% ACN) to give PNA dimer 7c (8.5 mg, 32%). Tr-HPLC:

5.23 (s, 2H, H_8'of G), 4.58 (s, 2H, H_8'of C), 4.14 (s, 1H, H_5'of G), 4.11 15.9 min (98%, 0-100% TEAB (0.05 M) ACN/H₂O 50/50 in 45 min).

(s, 1H, H_5'of C), 3.90 (m, 2H, H_10'), 3.86 (t, 2H, J=5.3 Hz, H_2'of G), ¹H NMR (major conformer, 400 MHz, 298 K, 8.3 mM, D₂O) (ppm):

3.70 (m, 2H, H_3'of G), 3.65 (m, 2H, H_3'of C), 3.51 (t, 2H, J=5.3 Hz ,H_2'8.12 (s, 1H, H₂of A), 8.02 (s, 1H, H₈of A), 6.89 (d, 1H, J=7.3 Hz, H₆of

of C). ¹³C{¹H} NMR major conformer (100 MHz, 323K, 15 mM, D₂O) C), 5.52 (d, 1H, J=7.3 Hz, H₅of C), 5.42 (s, 2H, H_8'of A), 4.44 (s, 2H,

(ppm): 176.7 (C_11', C), 174.2 (C_6'of G, C), 173.8 (C_6'of C, C), 172.8 H_8'of C), 4.14 (s, 1H, H_5'of A), 4.10 (s, 1H, H_5'of C), 3.89 (m, 2H,

(C_7'of C, C), 172.1 (C_7'of G, C), 169.3 (C₄of C, C), 160.5 (C₂of C, C), H_10'), 3.88 (m, 2H, H_2'of A), 3.75 (m, 2H, J=5.2 Hz, H_3'of A), 3.64 (m,

154.4 (C₄of G, C), 149.7 (C₆of C, CH), 143.0 (C₈of G, CH), 118.4 (C₅2H, H_3'of C), 3.51 (m, 2H, H_2'of C). ¹³C{¹H} NMR (major conformer,

of G, C), 97.7 (C₅of C, CH), 61.2 (C_2'of G, CH₂), 53.2 (C_3'of G, CH₂), 100 MHz, 298 K, 8.3 mM, D₂O) (ppm): 175.6 (C_11', C), 172.9 (C_6'of

52.6 (C_5'of G, CH₂), 52.3, (C_8'of C, CH₂), 51.9 (C_5'of C, CH₂), 50.7 (C_3'A, C), 172.2 (C_6'of C, C), 171.4 (C_7'of C, C), 170.8 (C_7'of A, C), 167.9

of C, CH₂), 47.5 (C_8'of G, CH₂), 44.7 (C_10', CH₂), 39.6 (C_2'of C, CH₂). (C₄of C, C), 159.3 (C₂of C, C), 156.8 (C₆of A, C), 153.7 (C₂of A, CH),

HRMS ESI⁺calcd for C₂₃H₃₂N₁₃O₈⁺(M+H)⁺618.2497, found 618.2494.

150.6 (C₄of A, C), 148.0 (C₆of C, CH), 144.3 (C₈of A, CH), 119.5 (C₅

of A, C), 96.5 (C₅of C, CH), 60.1 (C_2'of A, CH₂), 52.3 (C_3'of A, CH₂),

51.6 (C_5'of A, CH₂), 51.1, (C_8'of C, CH₂), 50.7 (C_5'of C, CH₂), 49.7 (C_3'

of C, CH₂), 46.7 (C_8'of A, CH₂), 43.5 (C_10', CH₂), 38.3 (C_2'of C, CH₂).

HRMS ESI⁺calcd for C₂₃H₃₂N₁₃O₇⁺(M+H)⁺602.2543, found 602.2542.

2-[((Guanin-9-yl)acetyl)-(2-hydroxyethyl)]amino-N-[2-[((Uracil-1-

yl)acetyl)-ethyl))]amino-N-[(carbamoyl)glycinamidyl]acetamide

(7b)

PNA dimer 6b (30 mg) was dissolved in THF/MeOH 1:3 (v:v) (1.2 mL)

and 28% aqueous ammonia was added (1.8 mL) The flask was 2-[((Thymin-9-yl)acetyl)-(2-hydroxyethyl)]amino-N-[2-[((thymin-1-

sealed and the mixture was stirred 6 h at 50 °C. The solvents were yl)acetyl)-ethyl))]amino-N-[(carboxyl)glycinamidyl]acetamide (7e).

then removed under reduced pressure and the crude product was PNA dimer 6e (50 mg) was dissolved in THF (3 mL) and TBAF 1 M in

taken up in THF (1.3 mL). A solution of TBAF (1 M) in THF (205 μL) THF (440 μL) was added. The reaction mixture was stirred 1 h at

was added and the reaction mixture was stirred 1 h at room room temperature. The solvent was removed under reduced

temperature. The reaction mixture was concentrated to dryness pressure and the crude material was dissolved in MeOH (2.5 mL),

under reduced pressure and the residue was purified by reverse NaOH 0.2 N (2.5 mL) was added and the reaction mixture was

phase column chromatography (0 to 100% of 0.05 M TEAB (pH 7.5) stirred 1 h at rt. The solution was neutralized with HCl 0.2 N. The

in 50% ACN) to give PNA dimer 7b (4,7 mg, 23% yield). Tr-HPLC: reaction mixture was concentrated to dryness under reduced

18.2 min (> 95%, 0-100% TEAB (0.05 M) ACN/H₂O 50/50 in 45 min). pressure and the residue was purified by reverse phase column

¹H NMR major conformer (600 MHz, 323 K, D₂O) (ppm): 7.68 (s, chromatography (0 to 100% of 0.05M TEAB buffer (pH 7.5) in 50%

1H, H₈of G), 7.27 (d, 1H, J=7.8 Hz, H₆of U), 5.60 (d, 1H, J=7.8 Hz, H₅ACN) to give PNA dimer 7e (15 mg, 45% yield). Tr-HPLC: 18.8 min

of U), 5.21 (s, 2H, H_8’of G), 4.66 (s, 2H, H_8’of U), 4.13 (s, 2H, H_5’of (99%, 0-100% TEAB (0.05M) ACN/H₂O 50/50 in 45 min). ¹H NMR

G), 4.12 (s, 2H, H_5’of U), 3.90 (s, 2H, H_10’), 3.85 (m, 2H, H_2’of G), (400 MHz, 298 K, 27 mM, D₂O) (ppm): Conformer 1 (major): 7.48

3.70 (m, 2H, H_3’of G), 3.61 (t, J=5.3 Hz, 2H, H_3’of U), 3.50 (t, J=5.3 (q, 1H, J=1.2 Hz, H₆of T₂), 7.19 (q, 1H, J=1.2 Hz, H₆of T₁), 4.86 (ABq,

Hz, 2H, H_2’of U). ¹³C{¹H} NMR major conformer (150 MHz, 323 K, 2H, ꢀν=7.0 Hz, H_8’of T₁), 4.65 (ABq, 2H, ꢀν=6.8 Hz, H_8’of T₂), 4.16

D₂O) (ppm): 176.9 (C_11’, CO), 174.3 (C_6’of G, CO), 173.8 (C_6’of U, (s, 2H, H_5’of T₂), 4.15 (s, 2H, H_5’of T₁), 3.93 (s, 2H, H_10’), 3.81 (t, 2H,

CO), 172.1 (C_7’of G, CO & C_7’of U, CO), 169.1 (C₄of U, CO), 161.3 & J=5.2 Hz, H_2’of T₁), 3.63 (t, 2H, J=5.2 Hz, H_3’of T₁), 3.59 (m, 2H, H_3’

156.5 (C₂of G, C & C₆of G, CO), 154.7 (C₄of G, C & C₂of U, CO), of T₂), 3.51 (m, 2H, H_2’of T₂), 1.76 (d, 3H, J=1.0 Hz, H₇of T₂), 1.75 (d,

150.0 (C₆of U, CH), 143.4 (C₈of G, CH), 118.4 (C₅of G, C), 103.9 (C₅3H, J=1.0 Hz, H₇of T₁). Conformer 2 : 7.41 (q, 1H, J=1.2 Hz, H₆of T₂),

of U, CH), 61.5 (C_2’of G, CH₂), 53.5 (C_3’of G, CH₂), 53.0 (C_5’of G, 7.40 (q, 1H, J=1.2 Hz, H₆of T₁), 4.88 (ABq, 2H, H_8’of T₁), 4.61 (ABq,

CH₂), 52.2 (C_5’of U, CH₂), 51.5 (C_8’of U, CH₂), 50.8 (C_3’of U, CH₂), ꢀν=7.2 Hz, 2H, H_8’of T₂), 4.30 (s, 2H, H_5’of T₂), 4.06 (s, 2H, H_5’of T₁),

47.4 (C_8’of G, CH₂), 44.9 (C_10’, CH₂), 39.8 (C_2’of U, CH₂). HRMS ESI⁺3.97 (s, 2H, H_10’), 3.78 (t, 2H, J=5.2 Hz, H_2’of T₁), 3.58 (m, 2H, H_3’of

calcd for C₂₃H₃₁N₁₂O₉⁺(M+H)⁺619.2337, found 619.2332.

T₁), 3.58 (m, 2H, H_3’of T₂), 3.43 (m, 2H, H_2’of T₂), 1.84 (d, 3H, J=1.0

Hz, H7 of T₂), 1.82 (d, 3H, J=1.0 Hz, H₇of T₁). ¹³C{¹H} NMR (major

conformer, 100 MHz, 298 K, 27 mM, D₂O) (ppm): 177.3 (C_11’, CO),

173.8 (C_6’of T₁, CO), 173.7 (C_6’of T₂, CO), 172.4 (C_7’of T₁, CO), 171.6

J. Name., 2013, 00, 1-3 | 18

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(C_7’of T₂, CO), 169.4 (C₄of T₂, C), 169.3 (C₄of T₁, C), 154.8 (C₂of T₁&

T₂, CO), 146.4 (C₆of T₁& T₂, CH), 113.0 (C₅of T₁, C), 112.7 (C₅of T₂,

C), 61.2 (C_2’of T₁, CH₂), 53.0 (C_3’of T₁, CH₂), 52.5 (C_5’of T₁, CH₂), 52.0

(C_8’of T₁, CH₂), 51.8 (C_8’of T₂, CH₂), 51.5 (C_5’of T₂, CH₂), 50.6 (C_3’of

T₂, CH₂), 44.7 (C_10’, CH₂), 39.1 (C_2'of T₂, CH₂), 13.9 (C₇of T₂, CH₃),

Acknowledgments

This work was supported by grant from the Aix-Marseille University.

We wish to thank Dr G. Valette (Montpellier University, France) for

his expertise in conducting mass spectrometry analysis. Ivan Barvik

was supported by the Czech Science Foundation under contract

#P205/12/G118.

+

13.7 (C₇of T₁, CH₃). HRMS ESI⁺calcd for C₂₄H₃₃N₈O₁₁(M+H)⁺

609.2269, found 609.2271.

Notes and references

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