mRNA Cap Modification through Carbamate Chemistry: Synthesis of Amino- and Carboxy-Functionalised Cap Analogues Suitable for Labelling and Bioconjugation

FULL PAPER

DOI: 10.1002/ejoc.201500672

mRNA Cap Modification through Carbamate Chemistry: Synthesis of Amino-

and Carboxy-Functionalised Cap Analogues Suitable for Labelling and

Bioconjugation

Marcin Warminski,^[^a^]^[^‡^]Zofia Warminska,^[^b^]^[^‡^]Joanna Kowalska,* and Jacek Jemielity*^[^b^]

[a]

Keywords: Nucleotides / mRNA / Conformation analysis / Fluorescence labeling / Biotinylation

A simple and efficient synthesis of dinucleotide mRNA 5Ј cap

analogues functionalised by the addition of an amine or carb-

oxylic acid group onto one of the ribose rings is reported.

Selection of the modification sites was dictated by the re-

cognition specificity for the 5Ј cap by various cap-binding

triphosphate bridge to confer resistance to specific hydro-

lases. Spatial crowding and structural rigidity were balanced

by use of diamines and amino acids of different lengths at-

tached to the ribose through a carbamate moiety. The impact

of these substitutions on the conformational equilibria of the

1

proteins, including eIF4E, DcpS, and Dcp2, as well as by bac- ribose components was investigated by H NMR spec-

teriophage RNA polymerase, which enables the incorpora-

troscopy. The utility of the analogues for labelling with fluo-

tion of the synthetic cap into the mRNA chain. Some ana- rescent dyes and affinity tags was demonstrated with the aid

logues were further modified by O-to-CH

2

substitution in the

of NHS activation chemistry.

Introduction

The eukaryotic mRNA cap is a structure present at the

[

1]

5Ј ends of all RNAs transcribed by RNA polymerase II.

A canonical mRNA cap consists of 7-methylguanosine

7

(

m G) linked to the first transcribed nucleotide through a

7

5

Ј,5Ј-triphosphate bridge (m GpppN, Figure 1), and is

7

often referred to as an m G cap. Because of its atypical

chemical structure, distinct from the rest of the RNA body,

the cap serves as a unique molecular anchor recognised by

several specialised proteins involved in the various stages of

mRNA maturation and turnover, including well-established Figure 1. Structure of the canonical mRNA 5Ј end.

functions in mRNA transport, translation, and degrada-

tion. Studies on these processes not only provide insight

into gene expression mechanisms, but may also contribute

to the design of new diagnostic applications or therapeutic

interventions.

[4]

used both as small ligands for structural, biophysical and

[5]

functional studies and as reagents for the preparation of

[6]

capped RNAs by in vitro transcription. However, recent

advancements in highly sensitive spectroscopic and molecu-

lar biology techniques, with detection limits down to the

single-molecule level, have opened up new possibilities for

research into cap-related processes, and thus provide plat-

forms for the continued development of more accurate and

efficient high-throughput assays. As such, new mRNA

Since its discovery nearly five decades ago,^[²^]studies of

the mRNA 5Ј cap and of cap-binding protein functions

have been significantly facilitated by the use of synthetic

[7]

7

mRNA cap analogues such as m G-containing mono-, di-

and oligonucleotides.^[Simple cap analogues have been

3]

[8]

cap analogues suitable for labelling with spectroscopic and

[

a] Division of Biophysics, Institute of Experimental Physics,

Faculty of Physics, University of Warsaw

Zwirki i Wigury 93, 02-089 Warsaw, Poland

E-mail: asia@biogeo.uw.edu.pl

biological tags by simple chemical methods would be of

[7a,9]

great utility.

Recently, several chemical and chemoen-

zymatic approaches to cap modification have been de-

[

10]

www.jemielitygroup.pl

scribed, for use in cap analogue immobilisation, fluores-

[

b] Centre of New Technologies, University of Warsaw,

Banacha 2c, 02-097 Warsaw, Poland

[11]

[12]

cence labelling

and bioconjugation.

For example, the

E-mail: jacekj@biogeo.uw.edu.pl

www.jemielitygroup.pl

Rentmeister group recently described several elegant meth-

7

ods to label the m G N position in the mRNA cap by

‡] These authors contributed equally to this work.

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201500672.

2

use of a recombinant Tgs1 N -methylase and synthetic S-

[

13]

adenosyl methionine analogues.

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However, chemical synthesis and modification of the cations in translational research and other cap-related

mRNA cap is not only a chemical challenge, due to the fields. As a result, here we report a general procedure for

7

sensitivity of m G to hydrolysis under acidic and alkaline the synthesis of functionalised cap analogues that offers

[

14]

conditions, but also a design challenge from the point of possibilities for structural optimisation with regard to the

view of maintaining the cap’s desired biological function linker position attachment, as well as its length, hydropho-

after conjugation with a molecular tag. For example, in the bicity and functional group. Taking advantage of carb-

context of co-transcriptional RNA capping and transla- amate chemistry and of coupling reactions mediated by di-

tional research, it is especially important for the mRNA cap valent metal chlorides, for ribose functionalisation and py-

to maintain interactions with two proteins: a bacteriophage rophosphate bond formation, respectively, we prepared a

7

RNA polymerase, which is responsible for incorporation of series of m GpppG derivatives that contained linkers at-

7

the cap onto the mRNA strand during in vitro transcrip- tached to the ribose moiety of either the first (m G) or the

tion, and the human translation initiation factor 4E second (G) nucleoside. The syntheses were exemplified by –

(

eIF4E), a cap-binding protein that recognises the cap dur- but are not limited to – cap analogues containing amino

2

ing translation initiation. It is well-established that the N

and carboxylic acid functional groups, which enabled the

7

position of m G plays a crucial role in the molecular re- subsequent attachment of carboxy- or amino-functionalised

cognition of the 5Ј cap by several cap-binding proteins, in- labels, respectively, through simple protocols employing N-

cluding eIF4E and decapping scavenger enzyme hydroxysuccinimide (NHS) esters generated in situ in water/

[

4c,4g]

2

(DcpS).

Consequently, modifications at the N posi- DMSO mixtures. Selected analogues were further modified

tion disrupt these interactions and are thus of limited use within the triphosphate bridge to confer enzymatic resis-

in functional studies of these proteins and their associated tance, which is beneficial for some biological applications.

processes. To address the demand for 5Ј cap analogues

suited to translational research, we recently reported on a

Results and Discussion

7

2

Ј-amino-substituted mRNA cap analogue (m G pppG)

N

that is amenable to subsequent biotinylation and incorpora-

The structures of cap analogues 1–13 synthesised in this

tion into the mRNA strand during in vitro transcription, work are shown in Figure 2. The analogues can be divided

7

subsequently yielding biotinylated mRNAs that are trans- into two main groups: those modified in the first (m G) or

lated in a cap-dependent manner and interact with streptav- the second (G) nucleoside. The linker introduced onto the

[

12a]

idin.

However, in a follow-up study we found that the ribose component of the guanosine moiety merely occupies

spatial crowding around the 2Ј-amino group impairs its the space where the RNA body attaches to the native cap

labelling reaction with bulkier tags. We thus envisaged that and, therefore, we expected that linkers at this position

the presence of a spacer separating the functional group would be accommodated by the majority of cap-binding

from the nucleoside moiety could facilitate the attachment proteins. As such, the guanosine-modified analogues (Fig-

of bulkier labels.

ure 2, Structure 1) should be useful as versatile substrates

Therefore, in this work we aimed at developing a simple for the preparation of labelled ligands for biophysical and

synthetic approach to create dinucleotide mRNA cap ana- biochemical experiments, preparation of affinity resins or

logues functionalised with linkers terminating in various bioconjugation to peptides and proteins. However, such an-

functional groups suitable for fluorescence labelling, bi- alogues would not be correctly incorporated into the

oconjugation or immobilisation and compatible with appli- mRNA 5Ј end by in vitro transcription reactions, which

Figure 2. Structures of the mRNA cap analogues synthesised in this work.

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Amino- and Carboxy-Functionalised mRNA Cap Analogues

should be initiated by attack of the cap’s guanosine 3Ј-OH tributylamine and repeated rotary evaporation from dry di-

group at the α-phosphate of the next template nucleotide. methylformamide (DMF). Treatment with 5 equivalents of

Therefore, for the purpose of RNA cotranscriptional modi- CDI in DMF led quantitatively to the 2Ј,3Ј-O,O-carbonate

[

17]

fication, we also synthesised analogues modified in the ri- of the nucleotide P-imidazolide. Prior to the addition of

7

bose component of the m G unit (Figure 2, Structure 2). a linker containing a primary amino group, excess CDI was

[

12a,15]

On the basis of several previous observations,

substi- decomposed by addition of anhydrous methanol. The

7

tutions on the ribose moiety of m G are expected to have amine group in the linker was then allowed to react with

little to no influence on the interaction between the mRNA the carbonate group to form a carbamate, as well as with

cap and eIF4E protein. As such, in-vitro-transcribed the substituted imidazole to form a phosphoramidate. The

capped mRNAs containing analogues modified in the ri- latter was hydrolysed under acidic conditions (pH 3.5) to

7

bose component of m G are expected to have translation yield a carbamoyl nucleotide derivative with an overall yield

efficiencies similar to those capped with standard ana- of 10–30%.

logues. Each group of analogues is represented by several

We tried to activate guanosine 5Ј-diphosphate (GDP) tri-

examples that differ in linker size and functional group. ethylammonium salt (15) by the literature protocol,^[but

These were synthesised as mixtures of two 2Ј-O and 3Ј-O omitting the laborious conversion into the tributylammon-

regioisomers (in 1:1–1:2 ratios), which could later be sepa- ium salt. However, the observed levels of conversion were

rated into pure regioisomers by RP-HPLC in the majority unsatisfactory, probably due to partial hydrolysis of CDI,

16]

of cases.

which is a moisture-sensitive reagent. Thus, we optimised

The performed syntheses are discussed in detail for ana- the reaction conditions by increasing the excess of CDI,

logues 1, 3, 4, 8 and 12 (Figure 2), because they are repre- changing the solvent to DMSO to improve GDP solubility

sentative examples for the various structures and synthetic and applying microwave heating to accelerate the reaction.

approaches used in this work and all contain the same Consequently, the GDP triethylammonium salt in DMSO

linker (L13_N= 4,7,10-trioxatridecane-1,13-diamine).

was treated with 10 equivalents of CDI and heated to 50 °C

The synthetic route to analogue 1, which contains a car- for 20 min with the aid of a microwave reactor (5 W) to

bamoyl moiety attached to guanosine, was based on the yield a 2Ј,3Ј-O,O-carbonylguanosine P-imidazolide quanti-

preparation of an appropriate modified guanine nucleotide tatively and instantly as determined by RP-HPLC (Fig-

(

18c, Scheme 1), and subsequent metal-chloride-mediated ure 3). Excess CDI was then readily hydrolysed before the

7

coupling with an imidazole-activated m G nucleotide (20, next step by the addition of 16 equivalents of water and

Scheme 3). The key intermediate 18c was obtained by treat- stirring until no gaseous CO was produced (approximately

2

ment of nucleotide with excess 1,1Ј-carbonyldiimidazole 15 min). As shown in Figure 3, the subsequent addition of

(CDI), and the subsequent opening of the resulting 2Ј,3Ј- 10 equivalents of 4,7,10-trioxatridecane-1,13-diamine and 5

O,O-cyclic carbonate with a primary amine by use of a pre- equivalents of 1,4-diazabicyclo[5.4.0]undec-7-ene (DBU) re-

viously described protocol,^[

16]

albeit with some crucial sulted in a nearly quantitative conversion of the cyclic carb-

modifications (Scheme 1). In the reported procedure, the onate into a mixture of 2Ј-O and 3Ј-O regioisomers of the

nucleotides were first converted into triethylammonium desired carbamate (R = 15.2 min and 16.2 min, respec-

t

salts by means of ion-exchange chromatography on Dowex tively) within 2 h. Negative electrospray ionisation mass

resin and then into tributylammonium salts by addition of spectrometry [ESI(–)MS] revealed that the two observed

Scheme 1. Synthesis of carbamoyl nucleotides 17a–b, 18a–f and 19.

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M. Warminski, Z. Warminska, J. Kowalska, J. Jemielity

FULL PAPER

products were P-imidazolides of carbamoylguanosine, dem- ciently stable to be isolated on preparative scale by ion-ex-

onstrating that the amine group reacts exclusively with the change chromatography. Consequently, we decided to hy-

carbonate moiety and does not substitute imidazole under drolyse it to the corresponding phosphate 18c under acidic

these conditions (Scheme 1), contrary to the mechanism conditions (pH 1; Scheme 1) before purification. After over-

suggested by Cremo et al.^[Notably, if excessive CDI hy- night stirring, RP-HPLC analysis revealed that two re-

drolysis before the amine addition was performed with an- gioisomers of 18c (L13 -GDP, R = 14.2 min and 14.4 min)

16]

N

t

[

16]

hydrous methanol according to the previous protocol,

were the only nucleotide compounds present in the reaction

the resulting dimethyl carbonate reacted with the added mixture (Figure 3). The product 18c (triethylammonium

amine to produce various byproducts, including nucleotide salt) was isolated as a mixture of regioisomers with the aid

derivatives with N-blocked amino groups (Figure S1 in the of DEAE-Sephadex and was used for further reactions in

Supporting Information).

that form after concentration. A sample was additionally

purified by semipreparative RP-HPLC to resolve the 2Ј-O

and 3Ј-O regioisomers for spectroscopic characterisation.

1

H NMR confirmed the positions of linker attachment and

enabled us to assign the isomers with shorter and longer

retention times as 2Ј-O-L13 -GDP and 3Ј-O-L13 -GDP,

N

respectively. On the basis of RP-HPLC analysis with UV/

Vis detection (Figure 3), we found that the 3Ј-O regioisomer

formed as the major product (ca. 1.5:1.0), which may be

due to the formation of a more stable 2Ј-O alkoxide anion

intermediate during the opening of the cyclic 2Ј,3Ј-O,O-

carbonate. Using essentially the same protocol, we also syn-

thesised GDP derivatives functionalised with ethylenedi-

amine (derivative 18a), hexamethylenediamine (derivative

18b), β-alanine (derivative 18d), γ-aminobutyric acid (deriv-

ative 18e), and 6-aminohexanoic acid (derivative 18f) with

68–85% yields.

The protocol of introducing a linker into the ribose ring

through a carbamate moiety (Scheme 1) has some limita-

tions resulting from the hydrolysis of P-imidazolides under

strongly acidic conditions. It is impossible either to obtain

carbamoyl nucleotides that are sensitive to such conditions

by this method, or to introduce linkers that are cleavable at

Figure 3. RP-HPLC reaction profiles from the synthesis of 18c with

use of unprotected guanosine diphosphate.

[

18]

low pH. Therefore, we developed an alternative synthesis

The obtained P-imidazolide in the crude form was not method for carbamoyl-functionalised nucleotides that in-

reactive in subsequent coupling reactions with 7-methyl- cluded protection of the terminal phosphate with a 2-cyan-

7

guanosine monophosphate (m GMP, 27) and not suffi- oethyl group to avoid the formation of a P-imidazolide moi-

Scheme 2. Synthesis of carbamoyl nucleotides 18a–f by use of a 2-cyanoethyl protecting group.

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Amino- and Carboxy-Functionalised mRNA Cap Analogues

ety (Scheme 2).^[¹⁹^]The appropriate phosphate-protected nu- 18c was isolated as a triethylammonium salt by DEAE-Se-

cleotide 26 was synthesised in a MgCl -mediated coupling phadex chromatography.

2

reaction between 2-cyanoethylphosphate P-imidazolide (25)

The purified 18c was then subjected to a zinc-chloride-

and guanosine monophosphate (14).^[Protection of the mediated coupling reaction with 7-methylguanosine P-

terminal phosphate elongates the phosphate chain by one imidazolide (20) to produce two isomers of cap analogue 1

residue; therefore, the method is generally limited to the as the major products (Scheme 3), which were then isolated

synthesis of functionalised nucleoside oligophosphates (di-, in 49% yield by DEAE-Sephadex ion-exchange chromatog-

tri- etc.). Introduction of the linker was performed as de- raphy. Unfortunately, the products eluted together with the

scribed for the unprotected nucleotides, with slight modifi- unreacted starting material 18c (ca. 25%) and thus required

cations. Treatment of 26 with CDI (10 equiv.) with micro- additional purification by semipreparative RP-HPLC with

wave heating (5 W, 50 °C, 5 min) produced the β-(2-cyano- use of triethylammonium acetate (TEAA) buffers. HPLC

ethyl)-diphosphate of 2Ј,3Ј-O,O-carbonylguanosine quanti- purification yielded the individual 2Ј-O and 3Ј-O regioiso-

tatively (Figure 4). Excess CDI was hydrolysed with water mers of 1 as triethylammonium salts.

20]

(18 equiv.), followed by addition of linker (5 equiv.) to form

Cap analogue 3 contains the same linker as 1 and an

additional methylenebisphosphonate modification in the

α,β-position of the triphosphate bridge. This product was

obtained by essentially the same procedure, except that

guanosine methylenebisphosphonate (16, Scheme 1) was

used as a starting material in place of GDP in the first step

Scheme 3). The final product 3 was isolated in 67%

yield.

The employment of a similar method to afford β,γ-meth-

a carbamate moiety. After the reaction was complete (≈2 h;

Figure 4), the 2-cyanoethyl protecting group was removed

by addition of DBU (10 equiv.) and heating the reaction

mixture to 50 °C under reduced pressure (to evaporate gen-

erated acrylonitrile) for 2–3 h (Figure 4). The reaction mix-

ture was then diluted with 10 volumes of 1% AcOH to

pH 6–7 and extracted with ethyl acetate, and the product

(

ylenebisphosphonate analogue 4 required the use of an un-

2

stable P -imidazolide of 7-methylguanosine methylene-

7

bisphosphonate (m GpCH p-Im), which proved to be not

2

very reactive in coupling reactions and susceptible to hy-

drolysis. Another possibility, the activation of carbamoyl-

guanosine monophosphate, failed in attempts to isolate this

compound, because we encountered the same problems as

described above for L13 -GDP-Im. Therefore, we devel-

N

oped an alternative one-pot synthesis starting from guan-

osine monophosphate (14) with a reversed order of linker

attachment and triphosphate bridge formation steps

(Scheme 4). In this approach, the intermediate compounds

were not isolated by column chromatography, which simpli-

fied the whole synthesis and ensured a good overall yield.

In the first step, GMP was activated with CDI (10 equiv.) in

DMSO to give 2Ј,3Ј-O,O-carbonylguanosine P-imidazolide.

Excess CDI was decomposed with water, followed by the

addition of 7-methylguanosine methylenebisphosphonate

(

21, 1 equivalent) and MgCl (16 equiv.). The reaction flask

2

was placed under reduced pressure for 15 min to remove

residual water, and the mixture was then stirred for a few

days until no further progress was observed by RP-HPLC.

Figure 4. RP-HPLC reaction profiles from the synthesis of 18c by

use of cyanoethyl-protected guanosine 5Ј-diphosphate.

Scheme 3. Synthesis of cap analogues 1–3 in coupling reactions between carbamoyl GDPs (18c, 18d and 19) and 7-methylguanosine 5Ј-

monophosphate P-imidazolide.

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Scheme 4. Synthesis of cap analogues 4–5 by a one-pot strategy.

8

Magnesium ions were complexed by addition of solid nucleophilic addition of a primary amine to the C position

Na EDTA (16 equiv.), and 4,7,10-trioxatridecane-1,13-di- is probably favoured over addition to the carbonate moiety

2

amine (8 equiv.) was added to give the final product 4. At because the electron-withdrawing effect of the carbonate

8

that stage, it was crucial to monitor the reaction progress group likely increased the electrophilic properties at the C

7

by RP-HPLC with UV/Vis and fluorescence detectors to position of m G. To verify this hypothesis, we performed

7

avoid a side-reaction leading to base-catalysed 7-methylgua- similar reactions with a mononucleotide m G derivative.

nosine imidazole ring opening.^[The ring opening reac- For that purpose, m GMP (27) was activated in situ with

14]

7

tion resulted in a byproduct that coeluted with the desired CDI, as described for GDP (15), and then 10 equivalents

product at R = 14.5 min but showed no fluorescence char- of 4,7,10-trioxatridecane-1,13-diamine were added. After

t

7

acteristic of m G (λ = 260 nm, λ_e_m= 370 nm), as indicated 10 min, the reaction mixture was analysed by RP-HPLC

ex

by a decreasing fluorescence to absorbance ratio of the ob- and tandem mass spectrometry. The two major products

served HPLC peak. Finally, compound 4 was isolated from lacked fluorescence, and fragmentation of the molecular

the reaction mixture as a triethylammonium salt by DEAE- ions (m/z = 894) yielded products of m/z = 386 in both

Sephadex column chromatography in a good overall yield cases. These correspond to 4,7,10-trioxatridecane-1,13-di-

of 20% (for all steps starting from 14).

amine connected to 7-methylguanine, and they fragmented

An analogous approach was tested for the synthesis of further to 7-methylguanine (m/z = 166) and 4,7,10-trioxatri-

analogue 12, functionalised in the ribose moiety of 7-meth- decane-1,13-diamine (m/z = 221; Figure S2 in the Support-

ylguanosine. For that purpose, we obtained an appropriate ing Information).

7

carbonate compound – 2Ј,3Ј-O,O-CO-m GppCH pG – in

Given these findings, we reasoned that it is necessary to

2

7

good yield (≈80% conversion by HPLC) by starting from introduce the linker prior to methylation of the N atom in

m GMP (27) and using a procedure analogous to that de- the guanosine moiety. We thus prepared carbamoyl-GMP

7

scribed for 4. Addition of the linker gave a product eluting 17a by the same procedure as described for 18c and treated

at R = 11.8 min with the expected m/z ratio by ESI(–)MS the isolated product (triethylammonium salt, 82% yield)

t

analysis (m/z = 1045), but lacking the characteristic fluores- with an excess (30 equiv.) of methyl iodide in DMSO to

7

cence for m G. Additionally, products with higher m/z val- obtain 22a (Scheme 5). Methylation proceeded selectively at

7

ues were also observed, suggestive of a double-linker ad- the N position without any signs of carbamate hydrolysis;

dition. During DEAE-Sephadex chromatography and sub- however, use of a prolonged reaction time resulted in a di-

7

sequent aqueous workup, the product with the expected m/z methylated (at N and phosphate) side product. We encoun-

ratio decomposed to a compound that eluted at R = tered some difficulties in isolating 22a because it does not

t

9.6 min and showed an m/z ratio of 1019, demonstrative of bind to standard anion-exchange resin (A25 DEAE Se-

the loss of a C=O moiety. In the solid isolated product, phadex) as a result of its very small net charge. Therefore,

the decomposition proceeded further, to afford a mixture of the reaction mixture was diluted with water and extracted

different products that were not further investigated. These with ethyl acetate to remove excess methyl iodide. The crude

observations suggested that the initial reaction with the product 22a remained in the aqueous phase and was used

8

amine had resulted in the formation of a C -substituted for coupling reactions after concentration without further

compound instead of the desired carbamate product. The purification.

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Amino- and Carboxy-Functionalised mRNA Cap Analogues

Scheme 5. Synthesis of cap analogues 6–13 by methylation of carbamoyl nucleotides and subsequent coupling either with guanosine

monophosphate P-imidazolide or with guanosine methylenebisphosphonate activated in situ.

The second reactant, guanosine methylenebisphosphon- DEAE-Sephadex isolation. The carbamoyl-7-methylguan-

ate P-imidazolide, was obtained in situ from 16, by use of osine derivative 23c was then coupled with 24 (1.2 equiv.)

CDI (10 equiv.) and microwave irradiation (5 W, 50 °C, in the presence of ZnCl (16 equiv.), and the final product

2

0 min). After the decomposition of excess CDI with water, 8 was isolated as a triethylammonium salt by DEAE-Se-

solutions of activated 16 (1.1 equiv.) and crude 22a (1 phadex purification in a 40% yield.

equivalent) were mixed, and ZnCl (16 equiv.) was added Cap analogues 6 and 7, containing diamine linkers of

Scheme 5). The coupling reaction was complete after ap- different lengths (ethylenediamine L2_Nand hexamethylene-

proximately 1 h, and the reaction mixture was diluted with diamine L6 , respectively), were synthesised and purified

2

(

N

16 equivalents of EDTA solution in 10 volumes of water according to the procedure described for 8. Interestingly,

alkalised to pH 9 with triethylamine to hydrolyse carbonate the difference in retention times for the 2Ј-O and 3Ј-O re-

selectively at the ribose unit of the second nucleoside gioisomers decreased with the shorter linkers and their sep-

(formed during the activation of 16). The product 12 was aration by semipreparative RP-HPLC was more challeng-

isolated as a triethylammonium salt by DEAE-Sephadex ing (for retention times see Exp. Sect.). Nevertheless, it was

chromatography to give a 33% yield starting from 17a.

still possible to obtain small product samples.

A similar synthetic scheme was applied for cap analogue

The synthesis of cap analogues 2, 5, 9, 10, 11 and 13,

8, which was obtained by coupling of 7-methylguanosine each containing a carboxy-functionalised linker, required

diphosphate 23c with guanosine monophosphate P-imidaz- only slight modifications of the procedures described for

olide 24 (Scheme 5), with some procedural modifications. the amino-functionalised cap analogues 1, 3, 4, 8 and 12.

Firstly, carbamoyl-GDP 18c was subjected to methylation To obtain the key carbamate-modified nucleotides 17b and

with methyl iodide; however, because of the limited solubil- 18d–f, commercially available amino acids in zwitterionic

ity of 18c in DMSO, the majority of the nucleotide re- form were used in reactions with 2Ј,3Ј-O,O-carbonate nu-

mained unreacted, and the yield after DEAE-Sephadex pu- cleotides. In order to deprotonate the amine groups and to

rification was Ͻ30%. To overcome this issue, we performed restore their nucleophilic properties, reactions were per-

the methylation reaction in aqueous solution at pH 4 with formed in the presence of equimolar amounts of DBU.

dimethyl sulfate, and obtained 23c with a satisfactory level Each of the carboxy-functionalised carbamoyl nucleotides

of conversion of 80% (Scheme 5) and a 74% yield after possessed an additional negative charge and associated tri-

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ethylammonium counterion, which increased its solubility

in DMSO and facilitated purification by ion-exchange

chromatography. As such, it was possible to perform the

methylation reaction with methyl iodide for both mono-

and diphosphates, with the resulting 7-methylguanosine de-

rivatives being easily isolated by DEAE-Sephadex even in

the case of monophosphate 22b, which was used as a pure

triethylammonium salt for the ZnCl -mediated coupling re-

action leading to 13.

2

Figure 5. Ribose ring pucker for 2Ј-O and 3Ј-O regioisomers of

carbamoyl 7-methylguanosine derivatives proposed on the basis of

Analogues 1–13 were obtained as mixtures of 2Ј-O/3Ј-O NMR studies.

regioisomers and isolated as triethylammonium salts. Small

aliquots of the final products and some of the intermediate

Finally, to verify the utility of our COOH- and NH₂-

compounds were chromatographed by semipreparative RP- functionalised cap analogues, we developed simple proto-

HPLC to separate regioisomers for spectroscopic character- cols for their conjugation with NH - and COOH-function-

2

1

isation. With the aid of the H NMR spectra of analogues alised labels, respectively, based on NHS-activation chemis-

6–13, we studied the influence of the linker attachment po- try commonly used for amide bond formation. For this pur-

sition on the conformational equilibrium [between two pose we selected analogue 12, containing an amino group,

stable conformers denoted N and S, or C3Ј-endo (3E) and analogue 10, containing a carboxyl group, and biotin and

C2Ј-endo (2E), respectively] of the 7-methylguanine ribose EDANS as the corresponding labels.

7

ring. The N conformer is preferred for m G in unmodified

Biotinylation of the 2Ј-O-regioisomer of analogue 12

7

+

cap analogues, such as m GpppG (65% N) and for the (NH₄salt) by use of NHS activation is shown in Scheme 6.

previously reported ARCA analogues (65% and 56% for An active NHS ester of biotin was first generated in situ by

7

,2Ј-O

7,3Ј-O

[6b]

m₂

GpppG and m₂

GpppG, respectively)

and is use of N,N,NЈ,NЈ-tetramethyl-O-(N-succinimidyl)uronium

[4b]

favoured for interactions with eIF4E.

Populations of tetrafluoroborate (TSTU, 1.1 equiv.) and Et N (2 equiv.) in

3

conformers N estimated from scalar coupling constants be- DMSO (to give a 0.4 m solution of biotin) and added por-

tween H1Ј and H2Ј^[are listed in Table 1, together with tionwise to a solution of the 2Ј-O-regioisomer of 12 in bor-

21]

7

7,2Ј-O/3Ј-O

the analogous data for m GpppG and m

GpppG ate buffer (pH 8.5). We determined that the reaction was

2

[

6b]

(ARCA).

The data suggest only a feeble influence of instantaneous and that prolonging the time of reaction had

linker length and its terminal functional group on the equi- no impact on the level of conversion. Complete conversion

librium of interest, but showed a marked difference in con- was achieved after addition of 3 equivalents of NHS ester

formation preference between the 2Ј-O and 3Ј-O regioiso- as monitored by RP-HPLC (Figure 6).

mers. In the case of 2Ј-O isomers, the N conformation for

The carboxy-functionalised cap analogue 10 (mixture of

7-methylguanosine is preferred (71–77% N, excluding 7). regioisomers and triethylammonium salt, Scheme 7) was

Conversely, the S conformation was preferred in 3Ј-O iso- also converted into its NHS ester in situ by use of TSTU

mers (39–49% N). In both cases the carbamoyl substituents (3 equiv.) and Et N (3 equiv.) in DMSO (to give a 0.05 m

3

adopt pseudo-axial positions; this minimises the steric hin- solution of the cap) and added to suspension of [5-(2-

drance between the linker and 7-methylguanine (Figure 5). aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS,

The conformational preference of 2Ј-O and 3Ј-O isomers 4 equiv.) in DMSO. The reaction progress was monitored

for N and S conformations, respectively, indicates that our with a RP-HPLC system equipped with an absorbance and

7

compounds may be useful for studying m G conforma- fluorescence detector to assess completion of the labelling

tional effects in interaction with cap-binding proteins.

reaction within 1 h.

1

7

Table 1. RP-HPLC retention times, H NMR chemical shifts for H1Ј of m G ribose, scalar coupling constants between H1Ј and H2Ј in

7

m G ribose and population of conformer N for m G ribose in 2Ј-O and 3Ј-O regioisomers of cap analogues 6–13.

Cap

analogue

Long name

RP-HPLC retention time^[^a^]Chemical shift of H1Ј

[min] [ppm]

Scalar coupling constant

J_H₁_Ј_,_H₂Ј [Hz]

Population of conformer

N (for m G) [%]

3

7

2Ј-O isomer 3Ј-O isomer 2Ј-O isomer 3Ј-O isomer 2Ј-O isomer 3Ј-O isomer 2Ј-O isomer 3Ј-O isomer

7

[b]

–

6

7

8

9

m GpppG

65

77

49

75

74

71

72

74

71

2

Ј/3Ј-O,7

56^[^b^]

49

45

m

2

GpppG

-m GpppG

-m GppCH

7

L2

L6

N

6.929

11.872

13.414

5.961

6.913

9.914

6.929

5.97

5.95

6.00

5.99

5.96

5.97

6.04

6.02

5.89

5.87

5.90

5.91

5.88

5.89

5.95

5.94

2.8

4.7

2.9

3.0

3.2

3.1

3.0

3.2

4.7

5.0

5.1

5.2

n.d.

5.4

4.9

5.4

7

N

11.975

13.738

6.208

7

L13

N

43

7

L2

C

L3

C

L5

C

42

7

[c]

n.d.^[^c^]

39

1

0

7.170

7

1

10.352

14.290

7.290

7

1

2

L13

L3

N

2

pG 13.960

pG 7.196

46

39

7

13

C

-m GppCH

2

[

a] Conditions described in the Supporting Information. [b] According to Jemielity et al.^[⁶^b^][c] Not determined because of the signal

shape.

6160

www.eurjoc.org

Eur. J. Org. Chem. 2015, 6153–6169

Amino- and Carboxy-Functionalised mRNA Cap Analogues

Scheme 6. Biotinylation of cap analogue 12 by use of in situ NHS

activation of d-biotin.

Scheme 7. Labelling of cap analogue 10 with EDANS by use of in

situ NHS activation of the carboxy-functionalised cap.

Figure 6. RP-HPLC profiles for the reaction between cap analogue

1

2 and the NHS ester generated in situ. The peak denoted with an

asterisk (*) with R

t

≈ 3 min is associated with the presence of

enebisphosphonate modifications to confer resistance to

Dcp2 and DcpS, respectively. For analogues 6–13, function-

alised in the ribose component of 7-methylguanosine, we

DMSO and TSTU in the reaction mixture and not a nucleotide

derivative.

1

performed H NMR conformational studies to examine the

In both cases, the products were subsequently isolated by

semipreparative RP-HPLC in 64 and 29% yield for 12-Biot

and 10-EDANS, respectively. The structures of the conju-

gates were confirmed by HRMS and MS/MS fragmentation

analysis. It is worth emphasising that despite the presence

of numerous functional groups in the cap structure, the

labelling reactions proceeded exclusively at the desired sites.

impact of the carbamoyl substitution on the conforma-

tional equilibrium of ribose. Finally, we successfully labelled

two representative analogues with biological (d-biotin) and

fluorescence (EDANS) tags. The choice of the functionali-

sation site was designed to maintain their unimpaired affin-

ity towards major cap-binding proteins. Therefore, our lab-

elled analogues are suitable as molecular probes for investi-

gation of cap-dependent processes. Moreover, the ability to

introduce a linker into the ribose component of 7-methyl-

guanosine creates the potential to incorporate analogues 6–

Conclusions

1

3 and their derivatives into mRNA through in vitro tran-

In this work we optimised the method of one-pot synthe-

sis of 2Ј-O/3Ј-O-carbamoyl nucleotides by use of unprotec-

ted or phosphate-protected nucleotides. By the first proto-

col, nine compounds were synthesised with yields ranging

from 68 to 85% and were later used for the preparation of

a series of 13 amino- or carboxy-functionalised mRNA 5Ј

scription in order to obtain site-specifically labelled

mRNAs. This is currently under investigation in our group.

Experimental Section

cap analogues, including three analogues containing α,β- General Information: Solvents, chemical reagents and starting mate-

methylenebisphosphonate and two containing β,γ-methyl- rials, including 14 (5Ј-GMP disodium salt), were from commercial

Eur. J. Org. Chem. 2015, 6153–6169

www.eurjoc.org

6161

M. Warminski, Z. Warminska, J. Kowalska, J. Jemielity

sources. Commercially available GMP disodium salt and 2-cyano- Emission spectra were recorded with a Perkin–Elmer LS-55 spec-

FULL PAPER

ethylphosphate barium salt were converted into triethylammonium

salts before synthesis. The conversion of GMP salt was performed

by passing an aqueous solution of GMP disodium salt through

Dowex 50W-X8 cationite resin in the triethylammonium form. The

conversion of 2-cyanoethylphosphate was performed by the pro-

In each case, the col-

lected eluate was concentrated under reduced pressure with re-

peated additions of ethanol, and the residue was dried in a vacuum

trofluorimeter set at excitation wavelengths of 280, 292 and 339 nm

with a 2.5 nm bandwidth and detection at 4.0 nm bandwidth.

Chemical Syntheses

Compounds 15, 16, 20, 21, 24, 25, 26 and 27 were synthesised by

the previously described protocols,^[

20,22]

with slight modifications.

_c_e_d_u_r_e_d_e_s_c_r_i_b_e_d_b_y_S_t_r_e_n_k_o_w_s_k_a_e_t_a_l_.[

19]

See the Supporting Information for the detailed protocols.

Procedure IA. Synthesis of the 2Ј-O/3Ј-O-Carbamoyl Nucleotides:

4

over P O10 to yield the triethylammonium salt as a white solid.

1,1Ј-Carbonyldiimidazole (CDI, 10 equiv.) was added to a solution

of 14 or 15 in DMSO (0.050 m), and the mixture was heated for

20 min in the microwave reactor with use of dynamic power mode

(Pmax = 5 W, Tmax = 50Ϯ1 °C). Then, excess CDI was hydrolysed

with water (15 equiv.), and linker (10 equiv.) was added, followed

by an equimolar amount of DBU. The reaction’s progress was mon-

itored by RP-HPLC. After completion of the linker attachment (1–

The synthesised nucleotides were purified by ion-exchange

–

chromatography on a DEAE Sephadex A-25 (HCO

3

form) col-

umn. For this, the column was loaded with the reaction mixture

and washed thoroughly with water until the eluate did not precipi-

tate with AgNO solution, in order to remove solvents and un-

3

bound reagents. Nucleotides were then eluted with use of a linear

gradient of triethylammonium hydrogen carbonate (TEAB) in de-

ionised water. Collected fractions were analysed spectrophotomet-

rically at 260 nm. Those containing the desired compound were

further analysed by RP-HPLC and combined. The yields were cal-

culated on the basis of optical density miliunits (mOD = ab-

sorbance of the solutionϫvolume in mL) of the isolated products

and corresponding starting materials (nucleotides or nucleotide P-

imidazolide derivatives). Optical unit measurements were per-

formed in phosphate buffer (pH 7.0, 0.1 m) at 260 nm for all nu-

2

h), the solution was diluted with hydrochloric acid (0.5%) until

pH 1 was reached, and the mixture was stirred overnight at room

temp. to hydrolyse the P-imidazolide completely. The neutralised

mixture was chromatographed on a DEAE-Sephadex column and

the eluate was concentrated to yield a triethylammonium salt of

the final product as a mixture of 2Ј-O/3Ј-O regioisomers.

L13

N

-GMP (17a): Compound 17a was synthesised according to

213 mg,

.428 mmol), CDI (694 mg) in DMSO (17 mL), water (116 μL),

,7,10-trioxatridecane-1,13-diamine (940 μL) and DBU (720 μL) to

Procedure IA, starting from 14 (5170 mOD260

0

4

,

7

cleotides, except for m G mononucleotide derivatives, for which

phosphate buffer (pH 6.0, 0.1 m) was used. After concentration un-

der reduced pressure with repeated additions of ethanol (96%) and

then acetonitrile (to decompose TEAB and to remove residual

water, respectively), nucleotides were isolated as triethylammonium

salts. The final compounds and several intermediate product

afford 17a (4440 mOD260, 3.34 g, 0.368 mmol, 82%), contaminated

with non-nucleotide compounds. The product was re-chromato-

graphed, yielding 3560 mOD260 (796 mg, 0.295 mmol) of still con-

taminated compound. A sample of 17a was additionally purified

and regioisomers were separated by semipreparative RP HPLC

with use of a linear gradient (3–18%) of acetonitrile in ammonium

2

Ј-O/3Ј-O regioisomers were separated by semipreparative RP-

HPLC with Discovery RP Amide C-16 HPLC column

25 cmϫ21.2 mm, 5-μm, flow rate 5.0 mLmin ) with UV detec-

a

t

acetate (pH 5.9, 0.05 m) over 240 min (R = 63.7 and 76.6 min for

–1

(

2Ј-O and 3Ј-O regioisomers, respectively).

tion at 254 nm. After repeated freeze-drying of the collected frac-

tions, single regioisomers were isolated as ammonium salts.

a

1

2

Ј-O Isomer: RP HPLC: R

t 2

= 15.5 min. H NMR (400 MHz, D O,

3

2

5 °C): δ = 8.13 (s, 1 H), 6.07 (d, JH,H = 5.7 Hz, 1 H), 5.53 (dd,

H,H = 5.7 Hz, 1 H), 4.67 (m, 1 H), 4.36 (m, 1 H), 4.11 (m, 2 H),

The structure and homogeneity of each final product were con-

firmed by further RP-HPLC, high-resolution mass spectrometry

3

J

3

.65–3.42 (m, 12 H), 3.24–3.06 (m, 4 H), 1.92 (m, 2 H, overlapped

1

using negative electrospray ionisation (HRMS-ESI) and H and

31

with residual acetate), 1.70 (m, 2 H) ppm. P NMR (162 MHz,

3

1

P NMR spectroscopy. Labelled cap analogues (10-EDANS and

2-Biot) were characterised by RP-HPLC and HRMS-ESI. Ad-

D

for C21

2

O, H

3 4

PO , 25 °C): δ = 1.8 (s, 1 P) ppm. HRMS ESI(–): calcd.

1

–

H

35

N

7

O

12

P

[M – H] 608.2087; found 608.20825.

ditional UV/Vis and fluorescence spectra were recorded for 10-ED-

ANS. The structures of side products were confirmed by low-reso-

a

1

3

2

Ј-O Isomer: RP HPLC: R

t

= 17.1 min. H NMR (400 MHz, D

2

O,

3

5 °C): δ = 8.13 (s, 1 H), 5.94 (d, JH,H = 7.2 Hz, 1 H), 5.33 (m, 1

H), 4.97 (m, JH,H = 7.2 Hz, 1 H), 4.45 (m, 1 H), 4.09 (m, 2 H),

.72–3.59 (m, 12 H), 3.25 (m, 2 H), 3.10 (m, 2 H), 1.95 (m, 2 H,

lution tandem mass spectrometry with negative and positive elec-

3

_t_r_o_s_p_r_a_y_i_o_n_i_s_a_t_i_o_n₍_E_S_I– and ESI+). Analytical HPLC was per-

3

formed with a Supelcosil LC-18-T HPLC column (4.6ϫ250 mm,

3

1

–1

overlapped with residual acetate), 1.81 (m, 2 H) ppm. P NMR

flow rate 1.3 mLmin ) and use of a linear gradient of 0–25% (con-

ditions “a”) or 0–50% (conditions “b”) of methanol in ammonium

acetate buffer (pH 5.9, 0.05 m) for 15 min and UV detection at

(

162 MHz, D

2

O, H

3

PO

4

, 25 °C): δ = 1.4 (s, 1 P) ppm. HRMS ESI-

–

(

–): calcd. for C21

H

35

N

7

O

12

P

[M – H] 608.2087; found

608.20824.

2

54 nm. Additional UV detection at 335 nm was applied for 10-

EDANS. Mass spectra were recorded with LTQ Orbitrap Velos

Thermo Scientific, high-resolution) and API 3200 Qtrap (AB-

Sciex, low-resolution) instruments. NMR spectra were recorded at

C

L3 -GMP (17b): Compound 17b was synthesised according to Pro-

cedure IA, starting from 14 (5170 mOD260, 213 mg, 0.428 mmol),

CDI (694 mg) in DMSO (17 mL), water (116 μL), γ-aminobutyric

(

1

2

5 °C with a Varian UNITY-plus spectrometer at 400 MHz ( H acid (441 mg) and DBU (720 μL) to afford 17b (4570 mOD260,

31

a

1

NMR) and 162 MHz ( P NMR), except for compound 8, which

was analysed at 501 and 203 MHz, respectively. H NMR chemical NMR (400 MHz, D

t

225 mg, 0.378 mmol, 85%). RP HPLC: R = 6.4 and 7.9 min. H

1

3

2

O, 25 °C): δ = 8.14 (br. s, 2 H), 6.07 (d, JH,H

3

shifts were calibrated to sodium 3-trimethylsilyl-[2,2,3,3-D

ionate (TSP) in D

O as an internal standard. ³¹P NMR chemical

shifts were calibrated to H PO (20%) in D O as an external stan-

4

]prop-

= 5.0 Hz, 1 H), 5.94 (d, JH,H = 7.2 Hz, 1 H), 5.48 (m, 1 H), 5.28

(m, 1 H), 4.94 (m, 1 H), 4.67 (m, 1 H), 4.46 (br. s, 1 H), 4.35 (br.

s, 1 H), 4.21–4.07 (m, 4 H), 3.14–3.00 (m, 4 H), 2.38 (m, 2 H), 2.28

2

3

4

2

3

1

dard. The raw NMR spectroscopic data were processed by use of

ACD/Labs 12.0 Software. Absorption spectra in the 700–220 nm

range were recorded with a Shimadzu UV-1800 instrument with

a 1.0 nm slit, 0.1 nm sampling interval, and medium scan speed.

(m, 2 H), 1.82 (m, 2 H), 1.70 (m, 2 H) ppm. P NMR (162 MHz,

D

2

O, H

for C15

491.09285.

3

PO

4

, 25 °C): δ = 0.39 (s, 2 P) ppm. HRMS ESI(–): calcd.

O P 491.0933 [M – H] ; found 491.09295 and

6 11

–

H

20

N

6162

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Eur. J. Org. Chem. 2015, 6153–6169

Amino- and Carboxy-Functionalised mRNA Cap Analogues

L2 -GDP (18a): Compound 18a was synthesised according to Pro-

cedure IA, starting from 15 (12010 mOD260, 650 mg, 0.994 mmol),

CDI (1.61 g) in DMSO (20 mL), water (270 μL) and ethylenedi-

–

N

1 P) ppm. HRMS ESI(–): calcd. for C21

688.1750; found 688.17374.

36 7 15 2

H N O P

[M – H]^–

C

L2 -GDP (18d): Compound 18d was synthesised according to Pro-

cedure IA, starting from 15 (990 mOD260, 51 mg, 0.082 mmol),

CDI (199 mg) in DMSO (3.3 mL), water (38 μL), β-alanine (73 mg)

amine (332 μL

.835 mmol, 84%). Regioisomers were separated by semiprepar-

ative RP HPLC with use of a linear gradient (0–15%) of aceto-

)

to afford 18a (10090 mOD260

,

526 mg,

0

and DBU (122 μL) to afford 18d (730 mOD260

.060 mmol, 74%). Regioisomers were separated by semiprepar-

ative RP HPLC with use of a linear gradient (0–15%) of aceto-

, 45.8 mg,

nitrile in ammonium acetate (pH 5.9, 0.05 m) over 180 min (R

t

=

0

24.0 and 26.6 min for 2Ј-O and 3Ј-O regioisomers, respectively).

2

Ј-O Isomer: RP HPLC: R^a

t

= 4.2 min. H NMR (400 MHz, D

1

2

O,

nitrile in ammonium acetate (pH 5.9, 0.05 m) over 480 min (R

30.9 and 33.1 min for 2Ј-O and 3Ј-O regioisomers, respectively).

t

=

3

2

5 °C): δ = 8.06 (s, 1 H), 6.06 (d, JH,H = 4.5 Hz, 1 H), 5.52 (dd,

H,H = 4.5 Hz, 1 H), 4.78–4.80 (m, 1 H, overlapped with water),

.35–4.20 (m, 3 H), 3.42 (t, JH,H = 5.7 Hz, 2 H), 3.11 (t, JH,H

.7 Hz, 2 H) ppm. P NMR (162 MHz, D

3

J

a

1

2

Ј-O Isomer: RP HPLC: R

t

= 4.2 min. H NMR (400 MHz, D

5 °C): δ = 8.17 (s, 1 H), 6.06 (d, JH,H = 4.2 Hz, 1 H), 5.48 (m, 1

H), 4.74 (m, 1 H), 4.43–4.17 (m, 3 H), 3.32 (m, 2 H), 2.44 (m, 2

2 3 4

H) ppm. P NMR (162 MHz, D O, H PO , 25 °C): δ = –9.82 (d,

2

O,

3

4

5

–

=

3

31

2

O, H

3

PO

4

, 25 °C): δ =

2

10.3 (d, JP,P = 20.6 Hz, 1 P), –11.1 (d, JP,P = 20.6 Hz, 1 P) ppm.

3

1

–

[M – H]^–528.0651;

HRMS ESI(–): calcd. for C13

found 528.06526.

H

20

N

7

O

12

P

2

J

P,P = 20.3 Hz, 1 P), –10.83 (d, JP,P = 20.3 Hz, 1 P) ppm. HRMS

–

ESI(–): calcd. for C14

557.04358.

H

19

N

6

O

14

P

2

[M – H] 557.0440; found

3

Ј-O Isomer: RP HPLC: R^a

t 2

= 4.5 min. H NMR (400 MHz, D O,

1

3

2

5 °C): δ = 8.10 (s, 1 H), 5.94 (d, JH,H = 6.7 Hz, 1 H), 5.38 (dd,

a

1

3

2

Ј-O Isomer: RP HPLC: R

t

= 4.6 min. H NMR (400 MHz, D

5 °C): δ = 8.14 (s, 1 H), 5.94 (d, JH,H = 7.5 Hz, 1 H), 5.33 (m, 1

H), 4.98 (m, 1 H), 4.48 (br. s, 1 H), 4.21 (m, 2 H), 3.39 (m, 2 H),

2

O,

3

J

H,H = 6.7 Hz, 1 H), 4.99 (dd, 1 H), 4.51 (m, 1 H), 4.22 (m, 2 H),

3

1

3

H

=

2

.50 (m, 2 H), 3.18 (m, 2 H) ppm. P NMR (162 MHz, D O,

2

3

PO

4

, 25 °C): δ = –10.5 (d, JP,P = 20.3 Hz, 1 P), –11.2 (d, JP,P

20.3 Hz, 1 P) ppm. HRMS ESI(–): calcd. for C13

3

1

2

–

.48 (m, 2 H) ppm. P NMR (162 MHz, D

2

O, H

3

PO

4

, 25 °C): δ =

–

20 7 12 2

H N O P

2

10.43 (d,

J

P,P = 20.3 Hz, 1 P), –11.15 (d,

J

P,P = 20.3 Hz, 1

–

[M – H] 528.0651; found 528.06503.

–

P) ppm. HRMS ESI(–): calcd. for C14

557.0440; found 557.04350.

H

19

N

6

O

14

P

2

[M – H]

N

L6 -GDP (18b): Compound 18b was synthesised according to Pro-

cedure IA, starting from 15 (990 mOD260, 51 mg, 0.082 mmol),

CDI (199 mg) in DMSO (3.3 mL), water (38 μL), hexamethylenedi-

C

L3 -GDP (18e): Compound 18e was synthesised according to Pro-

cedure IA, starting from 15 (9640 mOD260, 500 mg, 0.798 mmol),

CDI (1.29 g) in DMSO (32 mL), water (220 μL), γ-aminobutyric

amine (95 mg) and DBU (122 μL) to afford 18b (670 mOD260

,

a

3

8.4 mg, 0.055 mmol, 68%). RP HPLC: R

t

= 11.9 and 12.4 min.

acid (823 mg) and DBU (1.3 μL) to afford 18e (8154 mOD260

,

1

H NMR (400 MHz, D

2

O, 25 °C): δ = 8.13 (s, 1 H), 8.09 (s, 1 H),

a

1

452 mg, 0.675 mmol, 85%). RP HPLC: R

t

= 6.1 and 6.5 min. H

3

6

.05 (d, JH,H = 4.9 Hz, 1 H), 5.94 (d, JH,H = 7.2 Hz, 1 H), 5.52

m, 1 H), 5.32 (m, 1 H), 4.83–4.73 (m, 1 H, overlapped with water),

.99 (m, 1 H), 4.47 (br. s, 1 H), 4.35 (br. s, 1 H), 4.28–4.20 (m, 4

H), 3.11–2.91 (m, 8 H), 1.67 (m, 4 H), 1.54 (m, 4 H), 1.38 (m,

NMR (400 MHz, D

(

2

O, 25 °C): δ = 8.15 (s, 1 H), 8.10 (s, 1 H), 6.06

(

4

d, JH,H = 4.7 Hz, 1 H), 5.94 (d, ³JH,H = 7.0 Hz, 1 H), 5.50 (m, 1

3

H), 5.31 (m, 1 H), 4.99 (m, 1 H), 4.74 (m, 1 H, overlapped with

water), 4.48 (br. s, 1 H), 4.35 (br. s, 1 H), 4.29–4.17 (m, 4 H), 3.14–

31

6

H), 1.33–1.18 (m, 2 H, overlapped with TEA) ppm. P NMR

162 MHz, D O, H PO , 25 °C): δ = –8.81 (m, 2 P), –11.00 (br.

s, 2 P) ppm. HRMS ESI(–): calcd. for C17

3

.02 (m, 4 H), 2.40 (m, 2 H), 2.30 (m, 2 H), 1.82 (m, 2 H), 1.71

(

2

3

4

3

1

(

m, 2 H) ppm. P NMR (162 MHz, D

2

O, H

3 4

PO , 25 °C): δ =

–

28 7 12 2

H N O P [M – H]

–

C

10.71 (m, 2 P), –11.26 (br. s, 2 P) ppm. HRMS ESI(–): calcd. for

584.1277; found 584.12667 and 584.12620.

–

15

H

21

N

6

O

14

P

2

[M – H] 571.0596; found 571.06084 and

L13

N

-GDP (18c): Compound 18c was synthesised according to

480 mg,

.766 mmol), CDI (1.86 g) in DMSO (31 mL), water (0.36 mL),

,7,10-trioxatridecane-1,13-diamine (1.68 mL) and DBU (1.15 mL)

571.06079.

Procedure IA, starting from 15 (9250 mOD260

0

4

to afford 18c (6700 mOD260, 446 mg, 0.555 mmol, 72%). Regioiso-

mers were separated by semipreparative RP HPLC with use of a

linear gradient (3–18%) of acetonitrile in ammonium acetate

,

L5 -GDP (18f): Compound 18f was synthesised according to Pro-

C

cedure IA, starting from 15 (990 mOD260, 51 mg, 0.082 mmol),

CDI (199 mg) in DMSO (3.3 mL), water (38 μL), 6-aminohexanoic

acid (107 mg) and DBU (122 μL) to afford 18f (690 mOD260

,

a

4

1.8 mg, 0.057 mmol, 70%). RP HPLC: R

t

= 10.2 and 10.5 min.

O, 25 °C): δ = 8.18 (br. s, 2 H), 6.06 (d,

1

H NMR (400 MHz, D

³JH,H = 4.7 Hz, 1 H), 5.95 (d, JH,H = 7.2 Hz, 1 H), 5.50 (m, 1 H),

.31 (m, 1 H), 4.99 (m, 1 H), 4.72 (m, 1 H), 4.47 (br. s, 1 H), 4.36

2

(pH 5.9, 0.05 m) over 240 min (R

t

= 74.2 and 80.0 min for 2Ј-O and

3

3Ј-O regioisomers, respectively).

5

Ј-O Isomer: RP HPLC: R^a

= 14.9 min. H NMR (400 MHz, D

1

(br. s, 1 H), 4.28–4.18 (m, 4 H), 3.07 (m, 2 H), 2.98 (m, 2 H), 2.35

2

t

2

O,

3

2

J

5 °C): δ = 8.10 (s, 1 H), 6.07 (d, JH,H = 5.5 Hz, 1 H), 5.53 (dd,

H,H = 5.5 Hz, 1 H), 4.74 (m, 1 H, overlapped with water), 4.37

(m, 2 H), 2.30 (m, 2 H), 1.70–1.51 (m, 8 H), 1.45–1.33 (m, 4

3

31

2 3 4

H) ppm. P NMR (162 MHz, D O, H PO , 25 °C): δ = –11.19 (br.

–

(

2

m, 1 H), 4.25 (m, 2 H), 3.64–3.58 (m, 12 H), 3.25 (m, 2 H), 3.10 s, 4 P) ppm. HRMS ESI(–): calcd. for C17

m, 2 H), 1.95 (m, 2 H, overlapped with residual acetate), 1.81 (m, 599.0909; found 599.08957 and 599.09000.

H) ppm. ³¹P NMR (162 MHz, D

O, H PO , 25 °C): δ = –10.4

25 6 14 2

H N O P [M – H]

2

3

4

L13 -GpCH p (19): Compound 19 was synthesised according to

N

2

(m, 1 P), –11.2 (m, 1 P) ppm. HRMS ESI(–): calcd. for

Procedure IA, starting from 16 (13665 mOD260 728 mg,

.131 mmol), CDI (1835 mg, 10 equiv.) in DMSO (23 mL), water

(306 μL), 4,7,10-trioxatridecane-1,13-diamine (2.48 mL) and DBU

,

–

C

21

H

36

N

7

O

15

P

2

[M – H] 688.1750; found 688.17341.

1

Ј-O Isomer: RP HPLC: R^a

= 15.2 min. H NMR (400 MHz, D

1

3

t

2

O,

3

25 °C): δ = 8.13 (s, 1 H), 5.94 (d, JH,H = 7.2 Hz, 1 H), 5.33 (m, 1 (958 μL) to afford 19 [10370 mOD260, 3900 mg (contaminated with

3

H), 5.00 (m, JH,H = 7.2 Hz, 1 H), 4.48 (m, 1 H), 4.21 (m, 2 H), inorganic salts), 0.858 mmol, 76%]. Regioisomers were separated

3.72–3.59 (m, 10 H), 3.58–3.43 (m, 4 H), 3.22–3.11 (m, 2 H, over-

by semipreparative RP HPLC with use of a linear gradient (3–18%)

lapped with residual TEA), 1.93 (m, 2 H, overlapped with residual

of acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over 240 min

(R = 65.6 and 68.5 min for 2Ј-O and 3Ј-O regioisomers, respec-

t

tively).

31

acetate), 1.71 (m, 2 H) ppm. P NMR (162 MHz, D

2

O, H

3

PO

4

,

2

25 °C): δ = –10.3 (d, JP,P = 20.5 Hz, 1 P), –11.2 (d, JP,P = 20.5 Hz,

Eur. J. Org. Chem. 2015, 6153–6169

www.eurjoc.org

6163

M. Warminski, Z. Warminska, J. Kowalska, J. Jemielity

FULL PAPER

Ј-O Isomer: RP HPLC: R^a

= 14.4 min. H NMR (400 MHz, D

1

O,

0.259 mmol) and methyl iodide (480 μL, 30 equiv.) in DMSO

5 °C): δ = 8.13 (s, 1 H), 8.06 (d, JH,H = 5.5 Hz, 1 H), 5.54 (m, 1 (10 mL). The crude product was used for further reactions (see the

2

t

2

3

2

H), 4.74 (m, 1 H), 4.36 (m, 1 H), 4.22 (m, 2 H), 3.63 (m, 8 H),

synthesis of 12). An aliquot of 22a was isolated by semipreparative

2

3

.59–3.44 (m, 4 H), 3.25–3.07 (m, 4 H), 2.20 (t, JH,P = 20.0 Hz, 2 RP HPLC with use of a linear gradient (3–18%) of acetonitrile in

31

H), 1.93 (m, 2 H), 1.72 (m, 2 H) ppm. P NMR (162 MHz, D

H

2

O, ammonium acetate (pH 5.9, 0.05 m) over 180 min (R

, 25 °C): δ = 19.23 (m, 1 P), 15.72 (td, JH,P = 20.0, JP,P = 62.2 min for 2Ј-O and 3Ј-O regioisomers, respectively).

t

= 52.1 and

2

3

PO

4

–

9.5 Hz, 1 P) ppm. HRMS ESI(–): calcd. for C22

H

38

N

7

O

14

P

2

[M –

a

1

2

Ј-O Isomer: RP HPLC: R

t

= 15.4 min. H NMR (400 MHz, D

2

O,

–

H] 686.1957; found 686.19592.

3

5 °C): δ = 6.21 (d, JH,H = 4.0 Hz, 1 H), 5.50 (dd, JH,H = 4.0 Hz,

1

3

2

Ј-O Isomer: RP HPLC: R^a

t

= 14.9 min. H NMR (400 MHz, D

2

O,

1 H), 4.65 (dd, 1 H), 4.41 (m, 1 H), 4.16 (m, 1 H), 4.13 (s, 3 H),

3

5 °C): δ = 8.23 (s, 1 H), 5.94 (d, JH,H = 7.4 Hz, 1 H), 5.32 (m, 1 4.04 (m, 1 H), 3.70–3.61 (m, 10 H), 3.55 (m, 2 H), 3.19 (m, 2 H),

3

1

H), 5.02 (m, 1 H), 4.48 (m, 1 H), 4.18 (m, 2 H), 3.72–3.57 (m, 12 3.10 (m, 2 H), 1.95 (m, 2 H), 1.76 (m, 2 H) ppm. P NMR

3

H), 3.26 (m, 2 H), 3.11 (m, 2 H), 2.20 (t, JH,P = 20.0 Hz, 2 H), (162 MHz, D O, H PO , 25 °C): δ = 3.5 (s, 1 P) ppm. HRMS ESI-

2

3

4

.95 (m, 2 H), 1.82 (m, 2 H) ppm. ³¹P NMR (162 MHz, D

O, (–): calcd. for C H N O P

–

1

2

[M – H] 622.2243; found

2

37

7

12

H

3

PO

ESI(–): calcd. for C22

86.19595.

4

, 25 °C): δ = 19.08 (br. s, 1 P), 15.90 (m, 1 P) ppm. HRMS

622.22211.

–

[M – H]^–686.1957; found

H

38

N

7

O

14

P

2

a

t

1

3

2

1

Ј-O Isomer: RP HPLC: R

5 °C): δ = 6.12 (d, JH,H = 5.2 Hz, 1 H), 5.27 (dd, JH,H = 5.2 Hz,

H), 4.88 (dd, 1 H), 4.54 (m, 1 H), 4.15–4.10 (m, 5 H), 3.72–3.58

= 16.3 min. H NMR (400 MHz, D

2

O,

6

3

Procedure IB. Synthesis of the 2Ј-O/3Ј-O-Carbamoyl GDP: 1,1Ј-

Carbonyldiimidazole (CDI, 10 equiv.) was added to a solution of

(

m, 12 H), 3.24 (m, 2 H), 3.11 (m, 2 H), 1.95 (m, 2 H), 1.81 (m, 2

2

6 in DMSO (0.025 m) and the mixture was heated for 20 minutes

in the microwave reactor with use of dynamic power mode (Pmax

5 W, Tmax = 40Ϯ1 °C). The excess of CDI was then hydrolysed

31

H) ppm. P NMR (162 MHz, D

2

O, H

3

PO

4

, 25 °C): δ = 3.1 (s, 1

–

P) ppm. HRMS ESI(–): calcd. for C22

H

37

N

7

O

12

P

[M – H]

=

622.2243; found 622.22218.

with water (18 equiv.), and linker (5 equiv.) and DBU (5 equiv.)

were added. Progress of the reaction was monitored by RP HPLC.

After completion of the linker attachment (1–2 hours), more DBU

7

L3

C

-m GMP (22b): Compound 22b was synthesised according to

Procedure IIA, starting from 17b (4520 mOD260

.374 mmol) and methyl iodide (190 μL) in DMSO (15 mL) to af-

ford 22b (2190 mOD260, 113 mg, 0.192 mmol, 51%). RP HPLC: R

= 7.8 and 9.1 min. H NMR (400 MHz, D

m, 2 H), 6.21 (d, JH,H = 3.5 Hz, 1 H), 6.12 (d, JH,H = 4.7 Hz, 1

H), 5.51 (m, 1 H), 5.24 (m, 1 H), 4.87 (m, 1 H), 4.63 (m, 1 H), 4.57

br. s, 1 H), 4.43 (br. s, 1 H), 4.30–4.17 (m, 2 H), 4.12 (br. s, 8 H),

,

222 mg,

0

(10 equiv.) was added and the mixture was heated under reduced

pressure at 50 °C until complete removal of the 2-cyanoethyl pro-

tecting group. The mixture was diluted with acetic acid (0.5%, 10

volumes), neutralised, extracted with ethyl acetate and chromato-

graphed on DEAE-Sephadex. The eluate was concentrated to give

the triethylammonium salt of the final product as a mixture of 2Ј-

O/3Ј-O regioisomers.

a

1

t

2

O, 25 °C): δ = 9.18

3

(

3

(

3

1

.16–3.03 (m, 4 H), 2.28 (m, 4 H), 1.78 (m, 4 H) ppm. P NMR

O, H PO , 25 °C): δ = 0.32 (br. s, 2 P) ppm. HRMS

ESI(–): calcd. for C16

162 MHz, D

2

3

4

L2

N

-GDP (18a): Compound 18a was synthesised according to Pro-

–

H

22

N

6

O

11

P

[M – H] 505.1090; found

cedure IB, starting from 26 (5130 mOD260, 297 mg, 0.425 mmol),

CDI (690 mg) in DMSO (17 mL), water (138 μL), ethylenediamine

5

05.10798 and 505.10805.

7

L2

C

-m GDP (23d): Compound 23d was synthesised according to

30 mg,

.035 mmol) and methyl iodide (17 μL) in DMSO (1.4 mL) to af-

(

142 μL) and DBU (1.27 mL) with microwave heating to afford 18a

2940 mOD260, 141 mg, 0.243 mmol, 57%). Regioisomers were sep-

Procedure IIA, starting from 18d (420 mOD260

,

0

arated by semipreparative RP HPLC with use of a linear gradient

0–15%) of acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over

80 min (R = 24.0 and 26.6 min for 2Ј-O and 3Ј-O regioisomers,

respectively).

L13 -GDP (18c): Compound 18c was synthesised according to

Procedure IB, starting from 26 (5130 mOD260 297 mg,

.425 mmol), CDI (690 mg) in DMSO (17 mL), water (138 μL),

,7,10-trioxatridecane-1,13-diamine (465 μL) and DBU (1.27 mL)

ford 23d (220 mOD260, 11 mg, 0.019 mmol, 56%). Regioisomers

were separated by semipreparative RP HPLC with use of a linear

gradient (0–15%) of acetonitrile in ammonium acetate (pH 5.9,

.05 m) over 480 min (R

regioisomers, respectively).

(

1

t

0

t

= 29.6 and 32.9 min for 2Ј-O and 3Ј-O

N

,

a

1

2

1

t 2

Ј-O Isomer: RP HPLC: R = 5.3 min. H NMR (400 MHz, D O,

5 °C): δ = 6.19 (d, JH,H = 2.5 Hz, 1 H), 5.54 (m, 1 H), 4.69 (m,

H), 4.45–4.34 (m, 2 H), 4.23 (m, 1 H), 4.12 (s, 3 H), 3.34 (m, 2

2 3 4

H), 2.40 (m, 2 H) ppm. P NMR (162 MHz, D O, H PO , 25 °C):

δ = –9.74 (d, JP,P = 20.5 Hz, 1 P), –11.17 (d, JP,P = 20.5 Hz, 1

P) ppm. HRMS ESI(–): calcd. for C15

71.0596; found 571.05872.

0

4

3

with microwave heating to afford 18c (2830 mOD260, 185 mg,

.234 mmol, 55%). Regioisomers were separated by semiprepar-

ative RP HPLC with use of a linear gradient (3–18%) of aceto-

nitrile in ammonium acetate (pH 5.9, 0.05 m) over 240 min (R

4.2 and 80.0 min for 2Ј-O and 3Ј-O regioisomers, respectively).

3

1

0

2

–

H

21

N

6

O

14

P

2

[M – H]

t

=

5

7

a

1

Procedure IIA. Synthesis of the 2Ј-O/3Ј-O-Carbamoyl-m GMP and 3Ј-O Isomer: RP HPLC: R

2

t

= 6.2 min. H NMR (400 MHz, D

2

O,

7

3

Ј-O/3Ј-O-Carbamoyl-m GDP: Methyl iodide (8 equiv.) was added

25 °C): δ = 6.10 (d, JH,H = 5.7 Hz, 1 H), 5.28 (m, 1 H), 4.91 (m,

1 H), 4.58 (m, 1 H), 4.29 (m, 1 H), 4.19 (m, 1 H), 4.14 (s, 3 H),

3.36 (m, 2 H), 2.41 (m, 2 H) ppm. P NMR (162 MHz, D O,

2

3 4

H PO , 25 °C): δ = –7.83 (d, JP,P = 20.7 Hz, 1 P), –10.81 (d, JP,P

= 20.7 Hz, 1 P) ppm. HRMS ESI(–): calcd. for C15

[M – H] 571.0596; found 571.05971.

to a suspension of carbamoyl nucleotide (mixture of regioisomers)

in DMSO (0.025 mol of nucleotide per 1 L of solvent) and the

mixture was stirred at room temperature. The reaction’s progress

was monitored regularly by RP-HPLC to avoid phosphate methyl-

ation. The reaction was quenched by the addition of water and the

mixture was extracted three times with diethyl ether. The aqueous

3

1

2

–

21 6 14 2

H N O P

–

7

L3

C

-m GDP (23e): Compound 23e was synthesised according to

Procedure IIA, starting from 18e (8070 mOD260

.668 mmol) and methyl iodide (330 μL) in DMSO (27 mL) to af-

ford 23e (3740 mOD260, 192 mg, 0.328 mmol, 49%). Regioisomers

3

phase was neutralised with NaHCO and chromatographed on a

DEAE-Sephadex column. The eluate was then concentrated to

,

544 mg,

0

yield the triethylammonium salt of the final product.

7

L13

N

-m GMP (22a): Compound 22a was synthesised according to

were separated by semipreparative RP HPLC with use of a linear

Procedure IIA, starting from 17a (3130 mOD260

,

700 mg, gradient (0–15%) of acetonitrile in ammonium acetate (pH 5.9,

Eur. J. Org. Chem. 2015, 6153–6169

Amino- and Carboxy-Functionalised mRNA Cap Analogues

0

.05 m) over 480 min (R

t

= 43.3 and 52.1 min for 2Ј-O and 3Ј-O

(m, 1 H), 4.58 (m, 1 H), 4.46–4.19 (m, 5 H), 4.14 (s, 6 H), 3.18–

3.05 (m, 4 H), 2.99 (m, 4 H), 1.67 (m, 4 H), 1.52 (m, 4 H), 1.43–

regioisomers, respectively).

3

1

–

.30 (m, 8 H) ppm. P NMR (162 MHz, D

2

O, H

3

PO

4

, 25 °C): δ =

Ј-O Isomer: RP HPLC: R^a

= 6.6 min. H NMR (400 MHz, D

1

2

t

2

O,

2

8.89 (d,

11.12 (m, 2 P) ppm. HRMS ESI(–): calcd. for C18

JP,P = 21.5 Hz, 1 P), –9.18 (d, JP,P = 21.5 Hz, 1 P),

3

25 °C): δ = 9.23 (s, 1 H), 6.20 (d, JH,H = 3.0 Hz, 1 H), 5.53 (m, 1

–

30 7 12 2

H N O P

H), 4.69 (m, 1 H), 4.44–4.20 (m, 3 H), 4.13 (s, 3 H), 3.13 (m, 2 H),

.21 (m, 2 H), 1.75 (m, 2 H) ppm. ³¹P NMR (162 MHz, D

O,

PO , 25 °C): δ = –9.15 (br. s, 1 P), –10.97 (br. s, 1 P) ppm.

–

[M – H] 598.1433; found 598.14198 and 598.14165.

2

H

2

7

3

4

L13

N

-m GDP (23c): Compound 23c was synthesised according to

–

[M – H]^–585.0753; Procedure IIB, starting from 18c (6700 mOD260

HRMS ESI(–): calcd. for C16

found 585.07452.

H

23

N

6

O

14

P

2

,

446 mg,

0.555 mmol) and dimethyl sulfate (526 μL) in water (5.5 mL) to

afford 23c (4700 mOD260, 332 mg, 0.412 mmol, 74%). RP HPLC:

Ј-O Isomer: RP HPLC: R^a

5 °C): δ = 9.27 (s, 1 H), 6.12 (d, JH,H = 4.7 Hz, 1 H), 5.27 (br. s,

= 7.6 min. H NMR (400 MHz, D O,

1

3

2

1

t

2

a

1

R

6

t

= 14.7 and 15.2 min. H NMR (400 MHz, D

2

O, 25 °C): δ =

3

.20 (d, JH,H = 2.7 Hz, 1 H), 6.11 (d, ³JH,H = 5.0 Hz, 1 H), 5.55

3

H), 4.91 (br. s, 1 H), 4.59 (br. s, 1 H), 4.36–4.16 (m, 2 H), 4.13

(

m, 1 H), 5.28 (m, 1 H), 4.93 (m, 1 H), 4.72 (m, 1 H), 4.58 (m, 1

3

1

(s, 3 H), 3.17 (m, 2 H), 2.28 (m, 2 H), 1.78 (m, 2 H) ppm.

P

H), 4.44–4.19 (m, 5 H), 4.14 (m, 2 H), 3.72–3.56 (m, 24 H), 3.24

2 3 4

NMR (162 MHz, D O, H PO , 25 °C): δ = –10.26 (br. s, 2 P) ppm.

HRMS ESI(–): calcd. for C16

found 585.07463.

(

m, 4 H, overlapped with TEA), 3.11 (m, 4 H), 1.96 (m, 4 H), 1.80

m, 4 H) ppm. P NMR (162 MHz, D O, H PO , 25 °C): δ = –9.3

2 3 4

–

[M – H]^–585.0753;

H

23

N

6

O

14

P

2

3

1

(

m, 1 P), –11.0 (m, 1 P) ppm. HRMS ESI(–): calcd. for

7

–

L5

C

-m GDP (23f): Compound 23f was synthesised according to

15 2

C H N O P

[M

–

H] 702.1907; found 702.18909 and

2

38

7

Procedure IIA, starting from 18f (600 mOD260, 36 mg, 0.050 mmol)

702.18888.

and methyl iodide (25 μL) in DMSO (2.0 mL) to afford 23f

7

Procedure III. Synthesis of the m GppYpG-CO-Linker: Carbamoyl-

GpYp (mixture of regioisomers) and 20 (1.25 equiv.) were sus-

pended in DMF (to obtain a 0.05 m solution of carbamoyl-GpYp),

a

(

1

(

274 mOD260, 14 mg, 0.024 mmol, 48%). RP HPLC: R

t

= 10.2 and

1

2

0.5 min. H NMR (400 MHz, D O, 25 °C): δ = 9.27 (s, 1 H), 9.23

3

s, 1 H), 6.19 (d, JH,H = 3.5 Hz, 1 H), 6.11 (d, JH,H = 5.5 Hz, 1

2

and ZnCl (16 equiv.) was added. The mixture was vigorously

H), 5.52 (m, 1 H), 5.26 (m, 1 H), 4.90 (m, 1 H), 4.67 (m, 1 H), 4.58

stirred to dissolve the reagents. The reaction’s progress was moni-

tored by RP-HPLC. The reaction was quenched by addition of

EDTA (16 equiv.) solution in water, and the pH was adjusted to 6

with solid NaHCO . The resulting product was purified on a

3

DEAE-Sephadex column and isolated as a triethylammonium salt

after concentration of the eluate.

(

–

C

br. s, 1 H), 4.43 (br. s, 1 H), 4.40–4.16 (m, 4 H), 4.13 (s, 6 H), 3.08

m, 2 H), 2.98 (m, 2 H), 2.28 (m, 4 H), 1.71–1.47 (m, 8 H), 1.33

m, 4 H) ppm. ³¹P NMR (162 MHz, D

O, H

10.45 (m, 2 P), –11.24 (m, 2 P) ppm. HRMS ESI(–): calcd. for

2

3 4

PO , 25 °C): δ =

–

17

H

25

N

6

O

14

P

2

[M

–

H] 599.0909; found 599.08957 and

505.0900.

7

m GpppG-L13

N

(1): Compound 1 was synthesised according to

185 mg,

(511 mg) in DMF (4.6 mL)

7

Procedure IIB. Synthesis of the 2Ј-O/3Ј-O-Carbamoyl-m GDP:

Carbamoyl nucleotide (mixture of regioisomers) was dissolved in

water to obtain a 0.1 m solution, and the pH was adjusted to 4 with

acetic acid. Dimethyl sulfate (10 equiv.) was added in 10 aliquots

over 1 h. After each addition, the pH was readjusted to 4 by ad-

dition of NaOH (1 m). The reaction’s progress was monitored regu-

larly by RP-HPLC to avoid phosphate methylation. Reactions were

quenched by addition of NaOH to obtain a neutral pH and diluted

Procedure III, starting from 18c (2830 mOD260

.234 mmol), 20 (122 mg) and ZnCl

,

0

2

to afford 1 (2620 mOD260, 516 mg, 0.116 mmol, 49%), contami-

nated with 18c (25% of nucleotide content) and non-nucleotide

compounds. Product was purified and regioisomers were separated

by semipreparative RP HPLC with use of a linear gradient (3–18%)

of acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over 240 min

t

(R = 78.3 and 86.3 min for 2Ј-O and 3Ј-O regioisomers, respec-

10-fold with water. The mixture was extracted repeatedly with

tively).

chloroform and chromatographed on a DEAE-Sephadex column.

The eluate was then concentrated to yield the triethylammonium

salt of the final product.

a

1

2Ј-O Isomer: RP HPLC: R

t

= 15.2 min. H NMR (400 MHz, D

25 °C): δ = 9.04 (s, 1 H), 7.96 (s, 1 H), 5.92 (d, JH,H = 5.2 Hz, 1

H), 5.90 (d, JH,H = 3.7 Hz, 1 H), 5.40 (dd, JH,H = 5.2 Hz, 1 H),

.65 (dd, 1 H), 4.56 (dd, 1 H), 4.44 (dd, 1 H), 4.41–4.22 (m, 6 H),

.07 (s, 3 H), 3.65–3.41 (m, 12 H), 3.22–3.06 (m, 4 H), 1.93 (q, 2

H), 1.70 (m, 2 H) ppm. P NMR (162 MHz, D

δ = –11.2 (m, 2 P), –22.6 (m, 1 P) ppm. HRMS ESI(–): calcd. for

32 50 12 22 3

C H N O P [M – H] 1047.2381; found 1047.23743.

2

O,

3

7

L2

N

-m GDP (23a): Compound 23a was synthesised according to

4

Procedure IIB, starting from 18a (2750 mOD260

,

168 mg,

0.228 mmol) and dimethyl sulfate (220 μL) in water (2.3 mL) to

3

1

2 3 4

O, H PO , 25 °C):

afford 23a (274 mOD260, 14 mg, 0.024 mmol, 48%). RP HPLC: R

a

t

1

3

= 5.3 min. H NMR (400 MHz, D

2

O, 25 °C): δ = 6.20 (d, JH,H

–

3

=

2.0 Hz, 1 H), 6.11 (d, JH,H = 4.7 Hz, 1 H), 5.58 (m, 1 H), 5.31

a

1

(m, 1 H), 4.91 (m, 1 H), 4.83–4.74 (m, 1 H, overlapped with water),

3Ј-O Isomer: RP HPLC: R

t

= 15.5 min. H NMR (400 MHz, D

25 °C): δ = 8.01 (s, 1 H), 5.90 (d, JH,H = 3.5 Hz, 1 H), 5.81 (d,

JH,H = 7.0 Hz, 1 H), 5.27 (dd, 1 H), 4.89 (dd, 1 H), 4.55 (dd, 1

2

O,

3

4

3

.60 (m, 1 H), 4.42–4.20 (m, 5) 4.13 (s, 6 H), 3.56–3.40 (m, 4 H),

31

3

.20–3.13 (m, 2 H, overlapped with TEA) ppm.

P NMR

2

(162 MHz, D

2

O, H

3

PO

4

, 25 °C): δ = –8.69 (d, JP,P = 21.2 Hz, 1 H), 4.48–4.22 (m, 7 H), 4.03 (s, 3 H), 3.73–3.58 (m, 12 H), 3.24 (m,

2

31

P), –8.99 (d, JP,P = 22.0 Hz, 1 P), –11.03 (m, 2 P) ppm. HRMS 2 H), 3.12 (m, 2 H), 1.96 (q, 2 H), 1.81 (q, 2 H) ppm. P NMR

ESI(–): calcd. for C14

–

[M – H]^–542.0807; found

H

22

N

7

O

12

P

2

(162 MHz, D

2 3 4

O, H PO , 25 °C): δ = –11.2 (m, 2 P), –22.6 (m, 1

P) ppm. HRMS ESI(–): calcd. for C32

–

542.08064 and 542.08039.

H

50

N

12

O

22

P

3

[M – H]

1

047.2381; found 1047.23716.

7

L6

N

-m GDP (23b): Compound 23b was synthesised according to

7

Procedure IIB, starting from 18b (567 mOD260

.047 mmol) and dimethyl sulfate (45 μL) in water (500 μL) to af-

ford 23b (240 mOD260, 21 mg, 0.021 mmol, 45%). RP HPLC: R

11.9 and 12.4 min. H NMR (400 MHz, D

,

32 mg, m GpppG-L2

C

(2): Compound 2 was synthesised according to Pro-

-GDP, 2750 mOD260, 150 mg,

0.228 mmol), 20 (128 mg) and ZnCl (497 mg) in DMF (4.2 mL)

0

cedure III, starting from 18d (L2

C

a

t

2

1

=

(

2

O, 25 °C): δ = 9.27 to afford 2 (2130 mOD260, 96 mg, 0.094 mmol, 41%). Regioisomers

3

s, 1 H), 9.24 (s, 1 H), 6.20 (d, JH,H = 3.5 Hz, 1 H), 6.11 (d, JH,H

were separated for spectroscopic characterisation by semiprepar-

=

5.1 Hz, 1 H), 5.54 (m, 1 H), 5.27 (m, 1 H), 4.90 (m, 1 H), 4.68 ative RP HPLC with use of a linear gradient (0–15%) of aceto-

Eur. J. Org. Chem. 2015, 6153–6169

www.eurjoc.org

6165

M. Warminski, Z. Warminska, J. Kowalska, J. Jemielity

FULL PAPER

nitrile in ammonium acetate (pH 5.9, 0.05 m) over 420 min (R

a

1

t

=

2Ј-O Isomer: RP HPLC: R

t 2

= 14.9 min. H NMR (400 MHz, D O,

4

2.3 and 47.3 min for 2Ј-O and 3Ј-O regioisomers, respectively).

25 °C): δ = 9.32 (s, 1 H), 7.99 (s, 1 H), 5.95 (m, 2 H), 5.41 (dd,

JH,H = 3.0 Hz, 1 H), 4.69 (dd, 1 H), 4.62 (dd, 1 H), 4.51 (dd, 1

3

_Ј_-_O_I_s_o_m_e_r_:_R_P_H_P_L_C_:_Ra

1

2

t

= 5.9 min. H NMR (400 MHz, D

2

O,

3

H), 4.39–4.17 (m, 6 H), 4.08 (s, 3 H), 3.70–3.45 (m, 12 H), 3.19–

25 °C): δ = 9.02 (s, 1 H), 7.93 (s, 1 H), 5.90 (d, JH,H = 4.2 Hz, 1

3

3.07 (m, 4 H), 2.40 (t, JH,H = 20.4 Hz, 2 H), 1.93 (m, 2 H), 1.70

H), 5.88 (d, JH,H = 3.7 Hz, 1 H), 5.34 (m, 1 H), 4.65 (m, 1 H),

.55 (m, 1 H), 4.46–4.20 (m, 7 H), 4.07 (s, 3 H), 3.32 (m, 2 H), 2.46

(

m, 2 H) ppm. ³¹P NMR (162 MHz, D

2

O, H

3 4

PO , 25 °C): δ = 17.3

4

(

_m_,₂_H₎_p_p_m_.3 P NMR (162 MHz, D

1

(m, 1 P), 7.3 (m, 1 P), –11.0 (m, 1 P) ppm. HRMS ESI(–): calcd.

O, H PO , 25 °C): δ =

2 3 4

–

52 12 21 3

for C33H N O P [M – H] 1045.2588; found 1045.25662.

–11.40 (m, 2 P), –23.02 (m, 1 P) ppm. HRMS ESI(–): calcd. for

–

a

1

C

25

H

33

N

11

O

21

P

3

[M – H] 916.1701; found 916.10722.

3Ј-O Isomer: RP HPLC: R = 15.0 min. H NMR (400 MHz, D O,

t

2

3

Ј-O Isomer: RP HPLC: R^a

= 6.1 min. H NMR (400 MHz, D

1

O,

H,H

25 °C): δ = 9.28 (s, 1 H), 8.05 (s, 1 H), 5.94 (d, J = 3.2 Hz, 1

3

t

2

3

H), 5.83 (d, J

H,H

= 6.7 Hz, 1 H), 5.27 (m, 1 H), 4.91 (dd, 1 H),

25 °C): δ = 8.98 (s, 1 H), 8.02 (s, 1 H), 5.90 (d, JH,H = 3.5 Hz, 1

3

4.61 (dd, 1 H), 4.49 (m, 1 H), 4.46 (dd, 1 H), 4.38–4.17 (m, 5 H),

.06 (s, 3 H), 3.71–3.63 (m, 10 H), 3.60 (m, 2 H), 3.24 (m, 2 H),

H), 5.81 (d, JH,H = 7.0 Hz, 1 H), 5.26 (m, 1 H), 4.87 (m, 1 H),

.56 (m, 1 H), 4.49–4.21 (m, 7 H), 4.05 (s, 3 H), 3.39 (m, 2 H), 2.49

4

(

3

_m_,₂_H₎_p_p_m_.3 P NMR (162 MHz, D

1

3.11 (m, 2 H), 2.39 (t, J

H,H

= 20.3 Hz, 2 H), 1.95 (m, 2 H), 1.81

2

O, H

3

PO

4

, 25 °C): δ =

(

m, 2 H) ppm. ³¹P NMR (162 MHz, D

(m, 1 P), 7.3 (m, 1 P), –11.0 (m, 1 P) ppm. HRMS ESI(–): calcd.

2 3 4

O, H PO , 25 °C): δ = 17.3

–11.30 (br. s, 2 P), –22.63 (m, 1 P) ppm. HRMS ESI(–): calcd. for

–

C

25

H

33

N

11

O

21

P

3

[M – H] 916.1701; found 916.10792.

pG-L13 (3): Compound 2 was synthesised according

1500 mg,

(540 mg, 12 equiv.) in DMF

6.6 mL) to afford 3 (4960 mOD260, 230 mg, 0.220 mmol, 67%).

–

for C33

H

52

N

12

O

21

P

3

[M – H] 1045.2588; found 1045.25639.

(5): Compound 5 was synthesised according to

325 mg,

.633 mmol) and CDI (1.0 g) in DMSO (25 mL), water (183 μL),

1 (7220 mOD260, 351 mg, 0.633 mmol) and MgCl (964 mg) in

7

m GppCH

2

N

7

m GpCH

2

ppG-L2

C

to Procedure III, starting from 19 (3990 mOD260

.330 mmol), 20 (186 mg) and ZnCl

,

Procedure IV, starting from 14 (7650 mOD260

0

2

,

0

(

2

The product was purified for spectroscopic characterisation by se-

mipreparative RP HPLC with use of a linear gradient (3–18%) of

acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over 240 min (R

2

DMSO (another 5 mL), EDTA (3.77 g), β-alanine (564 mg) and

DBU (2.6 mL) to afford 5 (1060 mOD260, 48 mg, 0.047 mmol, 7%).

Regioisomers were separated for spectroscopic characterisation by

semipreparative RP HPLC with use of a linear gradient (0–15%)

of acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over 420 min

t

a

1

=

76.3 min). RP HPLC: R

t

= 15.0 min. H NMR (400 MHz, D

2

O,

25 °C): δ = 9.18 (s, 1 H), 9.16 (s, 1 H), 8.30 (s, 1 H), 8.25 (s, 1 H),

2

5

1

4

.98 (d, JH,H = 4.7 Hz, 1 H), 5.94 (m, 2 H), 5.86 (d, JH,H = 7.0 Hz,

H), 5.43 (m, 1 H), 5.27 (m, 1 H), 4.93 (m, 1 H), 4.69 (m, 1 H),

.60 (m, 2 H), 4.48 (m, 3 H), 4.42–4.21 (m, 11 H), 4.08 (s,1 H),

.06 (s, 1 H), 3.72–3.45 (m, 24 H), 3.24 (m, 2 H), 3.18–3.07 (m, 6

(

R

t

= 36.6 and 43.4 min for 2Ј-O and 3Ј-O regioisomers, respec-

tively).

a

1

2Ј-O Isomer: RP HPLC: R

t 2

= 6.2 min. H NMR (400 MHz, D O,

3

1

H), 2.42 (m, 4 H), 1.95 (m, 4 H), 1.85–1.68 (m, 4 H) ppm.

NMR (162 MHz, D

2

C

P

25 °C): δ = 9.31 (s, 1 H), 7.97 (s, 1 H), 5.94 (m, 2 H), 5.36 (m, 1

2

O, H

2

3

PO

4

, 25 °C): δ = 18.04 (m, 2 P), 8.50 (m,

H), 4.69 (m, 1 H), 4.61 (m, 1 H), 4.50 (m, 1 H), 4.40–4.17 (m, 6

P), –10.29 (m,

P) ppm. HRMS ESI(–): calcd. for

H), 4.08 (s, 3 H), 3.33 (m, 2 H), 2.48 (m, 2 H) 2.40 (m, 2 H) ppm.

–

33

H

52

N

12

O

21

P

3

[M – H] 1045.2588; found 1045.26040.

31

2 3 4

P NMR (162 MHz, D O, H PO , 25 °C): δ = 17.35 (br. s, 1 P),

7

Procedure IV. Synthesis of the m GpCH

2

ppG-CO-Linker: 1,1Ј- 7.26 (br. s, 1 P), –10.94 (m, 1 P) ppm. HRMS ESI(–): calcd. for

–

Carbonyldiimidazole (CDI, 10 equiv.) was added to a solution of

4 in DMSO (0.025 m) and the mixture was heated for 20 min in

the microwave reactor with use of dynamic power mode (Pmax

W, Tmax = 50Ϯ1 °C). The excess of CDI was then hydrolysed

with water (16 equiv.), and a solution (0.125 m) of 21 (1 equiv.) in

DMSO containing MgCl (16 equiv.) was added. The mixture was

26 35 11 20 3

C H N O P [M – H] 914.1278; found 914.12607.

1

a

1

3

2

Ј-O Isomer: RP HPLC: R

t

= 6.7 min. H NMR (400 MHz, D

2

O,

=

3

5 °C): δ = 9.29 (s, 1 H), 8.07 (s, 1 H), 5.94 (d, JH,H = 3.7 Hz, 1

H), 5.84 (d, JH,H = 7.0 Hz, 1 H), 5.27 (m, 1 H), 4.90 (m, 1 H),

5

3

4

.62 (m, 1 H), 4.50 (m, 1 H), 4.46 (br. s, 1 H), 4.38–4.16 (m, 5 H),

2

31

.07 (s, 3 H), 3.41 (m, 2 H), 2.56 (m, 2 H), 2.39 (m, 2 H) ppm.

P

stirred at room temp. until no further progress of the reaction was

observed on RP-HPLC chromatograms (a few days). EDTA (16 or

8

NMR (162 MHz, D

2

O, H PO , 25 °C): δ = 17.36 (br. s, 1 P), 7.21

3

4

(

C

br. s, 1 P), –11.07 (m, 1 P) ppm. HRMS ESI(–): calcd. for

equiv.) and linker (5 equiv.) were then added and the reaction’s

–

26

H

35

N

11

O

20

P

3

[M – H] 914.1278; found 914.12616.

progress was monitored by RP-HPLC to avoid nucleophilic ad-

ditions to the 7-methylguanosine imidazole ring. The reaction was

quenched by 10-times dilution with acetic acid (3%), and the re-

sulting mixture was chromatographed on a DEAE-Sephadex col-

umn. The eluate was then concentrated to yield the triethylammo-

nium salt of the final product as a mixture of 2ЈO/3ЈO regioisomers.

7

Procedure V. Synthesis of the Linker-CO-m GpppG: Carbamoyl-

7

m GDP (mixture of regioisomers) and 24 (1.25 equiv.) were sus-

pended in DMF (to obtain a 0.05 m solution of carbamoyl-

7

m GDP), and ZnCl

2

(16 equiv.) was added. The mixture was vigor-

ously stirred to dissolve the reagents, and the reaction’s progress

was monitored by RP-HPLC. After complete consumption of car-

m GpCH

2

ppG-L13

N

(4): Compound 4 was synthesised according

325 mg,

.633 mmol) and CDI (1.0 g) in DMSO (25 mL), water (183 μL),

1 (7220 mOD260, 351 mg, 0.633 mmol) and MgCl (964 mg) in

7

bamoyl-m GDP, the reaction was quenched by dilution (10-fold)

to Procedure IV, starting from 14 (7650 mOD260

0

2

,

with aqueous EDTA (16 equiv.) and the pH was adjusted to 6 with

solid NaHCO . The product was purified on DEAE-Sephadex and

3

isolated as a triethylammonium salt after evaporation of the eluate.

2

DMSO (another 5 mL), EDTA (1.88 g) and 4,7,10-trioxatridecane-

7

1

2

,13-diamine (690 μL) to afford 4 (2921 mOD260, 0.129 mmol,

0%), contaminated with 21 (48%). The product could be used for

L2

N

-m GpppG (6): Compound 6 was synthesised according to Pro-

cedure V, starting from 23a (970 mOD260, 66 mg, 0.085 mmol), 24

(45 mg), ZnCl (186 mg) in DMF (1.7 mL) to afford

(870 mOD260, 42 mg, 0.038 mmol, 45%), contaminated with 24

labelling without additional purification. An aliquot of 4 was puri-

fied and regioisomers were separated for spectroscopic characteri-

2

6

sation by semipreparative RP HPLC with use of a linear gradient (41%). Product could be used for labelling without additional puri-

3–18%) of acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over fication. An aliquot of 6 was purified and regioisomers were sepa-

(

2

40 min (R

t

= 68.3 and 70.9 min for 2Ј-O and 3Ј-O regioisomers,

rated for spectroscopic characterisation by semipreparative RP

HPLC with use of a linear gradient (0–15%) of acetonitrile in am-

respectively).

6166

www.eurjoc.org

Eur. J. Org. Chem. 2015, 6153–6169

Amino- and Carboxy-Functionalised mRNA Cap Analogues

monium acetate (pH 5.9, 0.05 m) over 480 min (R = 52.8 and

–

[M – H]^–

t

P) ppm. HRMS ESI(–): calcd. for C32

H

50

N

12

O

22

P

3

51.0 min for 2Ј-O and 3Ј-O regioisomers, respectively).

1047.2381; found 1047.23794.

Ј-O Isomer: RP HPLC: R^a

= 6.9 min. H NMR (400 MHz, D

1

3Ј-O Isomer: RP HPLC: R

25 °C): δ = 9.08 (s, 1 H), 7.99 (s, 1 H), 5.91 (d, JH,H = 5.1 Hz, 1

a

1

2

t

2

O,

t 2

= 13.7 min. H NMR (500 MHz, D O,

3

2

5 °C): δ = 8.00 (s, 1 H), 5.97 (d, JH,H = 2.5 Hz, 1 H), 5.78 (d,

3

J

H,H = 5.8 Hz, 1 H), 5.43 (dd, JH,H = 2.5, 4.9 Hz, 1 H), 4.67 (dd,

H), 5.79 (d, JH,H = 5.9 Hz, 1 H), 5.21 (dd, 1 H), 4.76 (dd, JH,H

³JH,H = 5.8 Hz, 1 H), 4.60 (dd, JH,H = 4.9 Hz, 1 H), 4.47 (dd, 1 = 5.1 Hz, 1 H, overlapped with water), 4.67 (dd, JH,H = 5.9 Hz, 1

3

H), 4.43–4.23 (m, 6 H), 4.05 (s, 3 H), 3.46 (m, 2 H), 3.15 (m, 2 H), 4.51 (m, 1 H), 4.46 (dd, 1 H), 4.40–4.20 (m, 5 H), 4.09 (s, 3

H) ppm. ³¹P NMR (162 MHz, D

O, H

11.7 (m, 2 P), –23.0 (t, 1 P) ppm. HRMS ESI(–): calcd. for H), 1.96 (m, 2 H), 1.81 (m, 2 H) ppm. P NMR (203 MHz, D

2

3 4

PO , 25 °C): δ = –11.3 to H), 3.72–3.65 (m, 10 H), 3.60 (m, 2 H), 3.23 (m, 2 H), 3.12 (m, 2

3

1

–

C

2

O,

3 4

H PO , 25 °C): δ = –11.3 (m, 2 P), –22.9 (m, 1 P) ppm. HRMS

–

24

H

34

N

12

O

19

P

3

[M – H] 887.1282; found 887.12663.

–

_Ј_-_O_I_s_o_m_e_r_:_R_P_H_P_L_C_:_Ra

1

ESI(–): calcd. for C32

1047.23732.

H

50

N

12

O

22

P

3

[M – H] 1047.2381; found

3

t

= 6.9 min. H NMR (400 MHz, D

2

O,

3

2

5 °C): δ = 9.02 (s, 1 H), 8.00 (s, 1 H), 5.89 (d, JH,H = 4.6 Hz, 1

3

7

H), 5.79 (d, JH,H = 6.0 Hz, 1 H), 5.22 (dd, 1 H), 4.75 (dd, JH,H

L2

C

-m GpppG (9): Compound 9 was synthesised according to Pro-

=

4.6 Hz, 1 H), 4.67 (dd, ³JH,H = 6.0 Hz, 1 H), 4.49 (m, 1 H), 4.46 cedure V, starting from 23d (1040 mOD260, 70 mg, 0.092 mmol), 24

dd, 1 H), 4.40–4.21 (m, 5 H), 4.07 (s, 3 H), 3.50 (m, 2 H), 3.18 (48 mg) and ZnCl (200 mg) in DMF (1.8 mL) to afford 9

(

2

m, 2 H) ppm. ³¹P NMR (162 MHz, D

m,

O, H

PO

, 25 °C): δ = –11.4

(850 mOD260, 38 mg, 0.037 mmol, 33%). Product was purified for

spectroscopic characterisation by semipreparative RP HPLC with

2

3

4

2

P), –23.0 (t, 1 P) ppm. HRMS ESI(–): calcd. for

–

C

24

H

34

N

12

O

19

P

3

[M – H] 887.1282; found 887.12653.

use of a linear gradient (0–15%) of acetonitrile in ammonium acet-

a

7

ate (pH 5.9, 0.05 m) over 420 min (R

t

= 37.5 min). RP HPLC: R

t

L6

cedure V, starting from 23b (1638 mOD260, 101 mg, 0.144 mmol),

4 (148 mg) and ZnCl (313 mg) in DMF (3.0 mL) to afford 7

N

-m GpppG (7): Compound 7 was synthesised according to Pro-

1

=

1

6.8 and 7.2 min. H NMR (400 MHz, D

H), 9.02 (s, 1 H), 5.99 (d, JH,H = 3.0 Hz, 1 H), 5.91 (d, JH,H

2

O, 25 °C): δ = 9.05 (s,

3

=

2

5

1

4

.2 Hz, 1 H), 5.82 (m, 2 H), 5.41 (m, 1 H), 5.18 (m, 1 H), 4.74 (m,

H, overlapped with water), 4.63 (m, 2 H), 4.51–4.15 (m, 15 H),

(1910 mOD260, 97 mg, 0.085 mmol, 59%), contaminated with 24

(35%). The product could be used for labelling without additional

3

1

.08 (s, 3 H), 4.06 (s, 3 H), 3.39 (m, 4 H), 2.53 (m, 4 H) ppm.

O, H PO , 25 °C): δ = –11.29 (br. s, 2 P),

22.80 (br. s, 1 P) ppm. HRMS ESI(–): calcd. for C25

P

purification. An aliquot of 7 was purified and regioisomers were

separated for spectroscopic characterisation by semipreparative RP

HPLC with use of a linear gradient (0–15%) of acetonitrile in am-

NMR (162 MHz, D

–

2

3

4

–

33 11 21 3

H N O P

–

[M – H] 916.1071; found 916.10641 and 916.10558.

monium acetate (pH 5.9, 0.05 m) over 180 min (R

t

= 79.7 and

7

81.7 min for 2Ј-O and 3Ј-O regioisomers, respectively).

L3

C

-m GpppG (10): Compound 10 was synthesised according to

_Ј_-_O_I_s_o_m_e_r_:_R_P_H_P_L_C_:_Ra

1

Procedure V, starting from 23e (2840 mOD260

,

138 mg,

2

t

= 11.9 min. H NMR (400 MHz, D O,

2

3

0.249 mmol), 24 (131 mg) and ZnCl (543 mg) in DMF (8.3 mL) to

2

J

5 °C): δ = 7.98 (s, 1 H), 5.95 (d, JH,H = 4.5 Hz, 1 H), 5.76 (d,

3

H,H ₌₆_.₀_H_z_,₁_H₎_,₅_.₃₀₍_d_d_,3

JH,H = 4.5 Hz, 1 H), 4.65 (dd,

afford 10 (4820 mOD260, 220 mg, 0.213 mmol, 68%). Regioisomers

were separated for spectroscopic characterisation by semiprepar-

ative RP HPLC with use of a linear gradient (0–15%) of aceto-

3_JH,H = 6.0 Hz, 1 H), 4.55 (dd, 1 H), 4.46 (dd, 1 H), 4.39–4.22 (m,

6

H), 4.07 (s, 3 H), 3.06–2.92 (m, 4 H), 1.57 (m, 2 H), 1.41–1.17

_m_,₆_H₎_p_p_m_.₃1P NMR (162 MHz, D

nitrile in ammonium acetate (pH 5.9, 0.05 m) over 480 min (R

t

=

(

2

O, H PO , 25 °C): δ = –10.7

3

4

59.8 and 62.3 min for 2Ј-O and 3Ј-O regioisomers, respectively).

m, 2 P), –22.2 (m, 1 P) ppm. HRMS ESI(–): calcd. for

–

a

1

C

28

H

42

N

12

O

19

P

3

[M – H] 943.1908; found 943.18859.

2Ј-O Isomer: RP HPLC: R

t

= 7.0 min. H NMR (400 MHz, D

2

O,

3

_Ј_-_O_I_s_o_m_e_r_:_R_P_H_P_L_C_:_Ra

1

25 °C): δ = 7.99 (s, 1 H), 5.96 (d, JH,H = 3.2 Hz, 1 H), 5.78 (d,

H,H = 6.0 Hz, 1 H), 5.39 (m, 1 H), 4.66 (m, 1 H), 4.59 (m, 1 H),

3

t

= 12.0 min. H NMR (400 MHz, D

2

O,

3

J

25 °C): δ = 7.98 (s, 1 H), 5.87 (d, JH,H = 5.0 Hz, 1 H), 5.77 (d,

3

4.48–4.21 (m, 7 H), 4.06 (s, 3 H), 3.11 (m, 2 H), 2.20 (m, 2 H), 1.73

J

H,H = 6.0 Hz, 1 H), 5.17 (dd, 1 H), 4.72–4.69 (m, JH,H = 4.6 Hz,

3

1

3

(m, 2 H) ppm. P NMR (162 MHz, D

2

O, H

3 4

PO , 25 °C): δ =

1

H, overlapped with water), 4.65 (dd, JH,H = 6.0 Hz, 1 H), 4.46

–

11.19 (br. s, 2 P), –22.74 (br. s, 1 P) ppm. HRMS ESI(–): calcd.

(

m, 2 H), 4.42–4.19 (m, 5 H), 4.08 (s, 3 H), 3.22–2.93 (m, 4 H),

–

_.₇₀_–₁_.₂₄₍_m_,₈_H₎_p_p_m_.3 P NMR (162 MHz, D

1

35 11 21 3

for C26H N O P [M – H] 930.1227; found 930.12044.

1

2

O, H

3 4

PO ,

a

1

2

5 °C): δ = –10.7 (m, 2 P), –22.2 (m, 1 P) ppm. HRMS ESI(–):

t 2

3Ј-O Isomer: RP HPLC: R = 7.2 min. H NMR (400 MHz, D O,

–

calcd. for C28

H

42

N

12

O

19

P

3

[M – H] 943.1908; found 943.18859.

25 °C): δ = 9.08 (s, 1 H), 7.98 (s, 1 H), 5.88 (br. s, 1 H), 5.78 (br. s,

7

1 H), 5.17 (br. s, 1 H), 4.73 (m, 1 H, overlapped with water), 4.66

L13

N

-m GpppG (8): Compound 8 was synthesised according to

(

D

br. s, 1 H), 4.48 (m, 2 H), 4.40–4.15 (m, 5 H), 4.08 (s, 3 H), 3.18

Procedure V, starting from 23c (1900 mOD260

.167 mmol), 24 (83 mg) and ZnCl (363 mg) in DMF (3.3 mL) to

,

134 mg,

3

1

m, 2 H), 2.36 (m, 2 H), 1.80 (m, 2 H) ppm. P NMR (162 MHz,

O, H PO , 25 °C): δ = –10.97 (br. s, 2 P), –22.73 (br. s, 1 P) ppm.

0

2

3

4

afford 8 (1500 mOD260, 71 mg, 0.066 mmol, 40%), contaminated

with 24 (16%). The product could be used for labelling without

additional purification. An aliquot of 8 was purified and regioiso-

mers were separated for spectroscopic characterisation by semipre-

parative RP HPLC with use of a linear gradient (3–18%) of aceto-

–

HRMS ESI(–): calcd. for C26

found 930.12192.

35 11 21 3

H N O P [M – H] 930.1227;

7

L5

C

-m GpppG (11): Compound 11 was synthesised according to

Procedure V, starting from 23f (170 mOD260, 11 mg, 0.015 mmol),

24 (7.8 mg) and ZnCl (32 mg) in DMF (830 μL) to afford 11

nitrile in ammonium acetate (pH 5.9, 0.05 m) over 240 min (R

t

=

2

6

0.7 and 63.3 min for 2Ј-O and 3Ј-O regioisomers, respectively).

(305 mOD260, 14 mg, 0.014 mmol, 73%). Regioisomers were sepa-

rated for spectroscopic characterisation by semipreparative RP

HPLC with use of a linear gradient (0–15%) of acetonitrile in am-

_Ј_-_O_I_s_o_m_e_r_:_R_P_H_P_L_C_:_Ra

1

2

t

= 13.4 min. H NMR (500 MHz, D

2

O,

3

2

J

5 °C): δ = 8.01 (s, 1 H), 6.00 (d, JH,H = 2.9 Hz, 1 H), 5.80 (d,

3

H,H ₌₅_.₉_H_z_,₁_H₎_,₅_.₄₂₍_d_d_,3

JH,H = 2.9 Hz, 1 H), 4.67 (dd,

t

monium acetate (pH 5.9, 0.05 m) over 240 min (R = 53.7 and

3_JH,H = 5.9 Hz, 1 H), 4.61 (dd, 1 H), 4.47 (dd, 1 H), 4.44–4.20 (m,

55.9 min for 2Ј-O and 3Ј-O regioisomers, respectively).

a

1

6

H), 4.08 (s, 3 H), 3.71–3.62 (m, 10 H), 3.57 (m, 2 H), 3.20 (m, 2 2Ј-O Isomer: RP HPLC: R

t

= 9.9 min. H NMR (400 MHz, D

2

O,

3

1

3

H), 3.11 (m, 2 H), 1.95 (m, 2 H), 1.78 (m, 2 H) ppm. P NMR 25 °C): δ = 9.04 (s, 1 H), 8.01 (s, 1 H), 5.97 (d, JH,H = 3.0 Hz, 1

3

(203 MHz, D

2

O, H

3

PO

4

, 25 °C): δ = –11.3 (m, 2 P), –22.6 (m, 1

H), 5.79 (d, JH,H = 6.0 Hz, 1 H), 5.38 (m, 1 H), 4.67 (m, 1 H),

Eur. J. Org. Chem. 2015, 6153–6169

M. Warminski, Z. Warminska, J. Kowalska, J. Jemielity

FULL PAPER

4.59 (m, 1 H), 4.47 (m, 1 H), 4.44–4.22 (m, 6 H), 4.07 (s, 3 H), 3.08 ZnCl

2

(386 mg, 2.83 mmol, 13.3 equiv.) and 22b (110 mg,

(

m, 2 H), 2.26 (m, 2 H), 1.57 (m, 2 H), 1.47 (m, 2 H), 1.30 (m, 2

0.177 mmol, 0.83 equiv.) were added. As the completion of cou-

pling reaction was achieved, the mixture was diluted with a solution

31

2 3 4

H) ppm. P NMR (162 MHz, D O, H PO , 25 °C): δ = –10.07 (br.

s, 2 P), –22.28 (br. s, 1 P) ppm. HRMS ESI(–): calcd. for

of EDTA (1.05 g, 2.83 mmol, 13.3 equiv.) and NaHCO

water (85 mL), and the pH was adjusted to 8.5 with triethylamine

ca. 1 mL) to hydrolyse the carbonate moiety. After overnight stir-

3

(0.53 g) in

–

C

28

H

39

N

11

O

21

P

3

[M – H] 958.1540; found 958.15379.

(

3

2

Ј-O Isomer: RP HPLC: R^a

t

= 10.3 min. H NMR (400 MHz, D

1

2

O,

ring the mixture was neutralised with acetic acid and chromato-

3

5 °C): δ = 9.08 (s, 1 H), 7.98 (s, 1 H), 5.90 (d, JH,H = 6.0 Hz, 1

+

graphed on DEAE-Sephadex, yielding 13 as a TEAH salt

3

H), 5.78 (d,

JH,H = 5.7 Hz, 1 H), 5.17 (m, 1 H), 4.73 (m, 1 H,

(

880 mOD260, 43 mg, 0.039 mmol, 22%). Regioisomers were sepa-

overlapped with water), 4.66 (m, 1 H), 4.49 (br. s, 1 H), 4.45 (m, 1

H), 4.39–4.17 (m, 5 H), 4.08 (s, 3 H), 3.13 (m, 2 H), 2.29 (m, 2 H),

rated for spectroscopic characterisation by semipreparative RP

HPLC with use of a linear gradient (0–15%) of acetonitrile in am-

monium acetate (pH 5.9, 0.05 m) over 360 min (R = 50.1 min and

t

1

.64–1.48 (m, 4 H), 1.35 (m, 2 H) ppm. ³¹P NMR (162 MHz, D

2

O,

H PO , 25 °C): δ = –10.66 (br. s, 2 P), –22.27 (br. s, 1 P) ppm.

3 4

54.4 min for 2Ј-O and 3Ј-O isomers respectively).

39 11 21 3

H N O P

[M – H]^–958.1540;

–

HRMS ESI(–): calcd. for C28

found 958.15393.

a

1

t 2

2Ј-O Isomer: RP HPLC: R = 7.3 min. H NMR (400 MHz, D O,

3

2

5 °C): δ = 9.16 (s, 1 H), 8.10 (s, 1 H), 6.02 (d, JH,H = 3.0 Hz, 1

H), 5.82 (d, JH,H = 5.7 Hz, 1 H), 5.43 (m, 1 H), 4.69 (m, 1 H),

.62 (m, 1 H), 4.49 (m, 1 H), 4.44–4.17 (m, 6 H), 4.08 (s, 3 H), 3.15

m, 2 H), 2.37 (m, 4 H), 1.77 (m, 2 H) ppm. P NMR (162 MHz,

O, H PO , 25 °C): δ = 17.20 (br. s, 1 P), 7.80 (br. s, 1 P), –11.16

m, P) ppm. HRMS ESI(–): calcd. for

7

L13

N

-m GppCH

2

pG (12): 1,1Ј-Carbonyldiimidazole (CDI, 373 mg,

3

2.30 mmol, 10 equiv.) was added to a solution (0.05 m) of 16

4

(

(191 mg, 0.230 mmol, 1 equiv.) in DMSO (5 mL) and the mixture

3

1

was heated for 20 min in the microwave reactor with use of dynamic

power mode (Pmax = 5 W, Tmax = 50Ϯ1 °C). The excess of CDI

was then hydrolysed with water (62 μL, 3.45 mmol, 15 equiv.), and

a crude solution of 22a (8 mL, 0.207 mmol, 0.9 equiv.) in DMSO

D

(

[

2

3

4

–

1

27 37 11 20 3

C H N O P

–

M – H] 928.1435; found 928.14130.

a

1

containing ZnCl

2

(502 mg, 3.68 mmol, 16 equiv.) was added. After

3Ј-O Isomer: RP HPLC: R

t

= 8.6 min. H NMR (400 MHz, D

2

O,

3

ca. 30 min, as the completion of the coupling reaction was

achieved, the mixture was diluted with a solution of EDTA (1.37 g,

25 °C): δ = 9.20 (s, 1 H), 8.03 (s, 1 H), 5.94 (d, JH,H = 5.5 Hz, 1

H), 5.78 (d, JH,H = 5.7 Hz, 1 H), 5.18 (m, 1 H), 4.80 (br. s, 1 H,

3

.68 mmol, 16 equiv.) and NaHCO

3

(0.68 g) in water and the pH

overlapped with water), 4.69 (m, 1 H), 4.53 (br. s, 1 H), 4.48 (m, 1

H), 4.36–3.17 (m, 5 H), 4.09 (s, 3 H), 3.17 (m, 2 H), 2.36 (m, 4 H),

was adjusted to 8.5 with triethylamine. After overnight stirring the

mixture was neutralised with acetic acid and chromatographed on

DEAE-Sephadex, yielding 12 as a TEAH salt (1550 mOD260

3

1

1.79 (m, 2 H) ppm. P NMR (162 MHz, D

2 3 4

O, H PO , 25 °C): δ =

+

,

17.21 (br. s, 1 P), 7.75 (m, 1 P), –11.07 (m, 1 P) ppm. HRMS ESI-

–

1.74 g, 0.069 mmol, 33% starting from 17a), contaminated with (–): calcd. for C27

H

37

N

11

O

20

P

3

[M – H] 928.1435; found

non-nucleotide compounds. The product was additionally purified

and regioisomers were separated for spectroscopic characterisation

by semipreparative RP HPLC with use of a linear gradient (3–18%)

of acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over 240 min

928.14098.

Synthesis of Labelled Cap Analogues

7

EDANS-L3 -m GpppG (10-EDANS): N,N,NЈ,NЈ-Tetramethyl-O-

(N-succinimidyl)uronium tetrafluoroborate (TSTU, 7.6 mg,

C

(R

t

= 63.3 min and 67.3 min for 2Ј-O and 3Ј-O isomers, respec-

tively).

25.3 μmol, 3 equiv.) and triethylamine (3.5 μL, 25.3 μmol, 3 equiv.)

was added to a solution of cap analogue 10 (8.0 mg, 8.4 μmol,

Ј-O Isomer: RP HPLC: R^a

= 14.0 min. H NMR (400 MHz, D

1

O,

2

t

2

1

3

equiv.) in DMSO (169 μL) and the mixture was vortexed for

0 min at room temperature. A solution of the activated cap ana-

3

25 °C): δ = 9.16 (s, 1 H), 8.17 (s, 1 H), 6.04 (d, JH,H = 3.0 Hz, 1

3

H), 5.83 (d, JH,H = 5.7 Hz, 1 H), 5.45 (dd, JH,H = 3.0, 5.0 Hz, 1

logue was added to a solution of [5-(2-aminoethyl)amino]naphthal-

ene-1-sulfonic acid (EDANS, 9.0 mg, 33.8 μmol, 4 equiv.) in

DMSO (169 μL). The reaction’s progress was monitored by RP-

HPLC with UV/Vis detection at 254 and 335 nm. After the reaction

was complete, the mixture was diluted with 10 volumes of water

and the product was isolated as an ammonium salt (3.1 mg,

3

H), 4.68 (dd, JH,H = 5.9 Hz, 1 H), 4.63 (dd, JH,H = 5.0 Hz, 1 H),

.49 (dd, 1 H), 4.43–4.18 (m, 6 H), 4.08 (s, 3 H), 3.70–3.62 (m, 10

4

3

H), 3.57 (m, 2 H), 3.19 (m, 2 H), 3.10 (m, 2 H), 2.39 (t, JH,H

0.5 Hz, 2 H), 1.95 (m, 2 H), 1.77 (m, 2 H) ppm. ³¹P NMR

162 MHz, D O, H PO , 25 °C): δ = 17.1 (m, 1 P), 7.9 (m, 1 P),

11.1 (m, 1 P) ppm. HRMS ESI(–): calcd. for C33

=

2

(

–

2

3

4

–

52 12 21 3

H N O P

2.4 μmol, 29%) by semipreparative RP-HPLC with use of a linear

–

[M – H] 1045.2588; found 1045.25657.

gradient (0–15%) of acetonitrile in ammonium acetate (pH 5.9,

Ј-O Isomer: RP HPLC: R^a

= 14.3 min. H NMR (400 MHz, D

1

O,

b

3

t

2

t t

0.05 m) over 120 min (R = 83.0 min). RP HPLC: R = 12.2 min.

3

–

25 °C): δ = 9.20 (s, 1 H), 8.15 (s, 1 H), 5.95 (d, JH,H = 5.0 Hz, 1 HRMS ESI(–): calcd. for C38H N O P S [M – H] 1187.1847;

47 13 23 3

3

H), 5.82 (d, JH,H = 5.5 Hz, 1 H), 5.21 (dd, 1 H), 4.81 (dd, JH,H

found 1187.18038.

3

=

5.0 Hz, 1 H, overlapped with water), 4.67 (dd, JH,H = 5.5 Hz, 1

7

Biot-L13

N

-m GppCH

2

pG (12-Biot): TSTU (5.0 mg, 16.5 μmol,

H), 4.52 (m, 1 H), 4.48 (dd, 1 H), 4.37–4.15 (m, 5 H), 4.08 (s, 3

H), 3.71–3.63 (m, 10 H), 3.59 (m, 2 H), 3.22 (m, 2 H), 3.11 (m, 2

4

equiv.) and triethylamine (2.2 μL, 16.5 μmol, 4 equiv.) were added

to a solution of d-biotin (4.0 mg, 16.5 μmol, 4 equiv.) in DMSO

42 μL) and the mixture was vortexed for 30 min at room tempera-

ture. A solution of the activated d-biotin was added portionwise

10 additions over 1 h) to a solution of cap analogue 12 (5.0 mg,

.1 μmol, 1 equiv.) in borate buffer (pH 8.5, 42 μL). The reaction’s

pG (13): 1,1Ј-Carbonyldiimidazole (CDI, 345 mg, progress was monitored by RP-HPLC and the pH was maintained

3

H), 2.39 (t,

J

H,H = 20.3 Hz, 2 H), 1.95 (m, 2 H), 1.79 (m, 2

O, H PO , 25 °C): δ = 17.2 (m,

P), 7.9 (m, 1 P), –11.0 (m, 1 P) ppm. HRMS ESI(–): calcd. for

(

H) ppm. ³¹P NMR (162 MHz, D

1

2

3

4

(

4

–

C

33 52 12 21 3

H N O P [M – H] 1045.2588; found 1045.25660.

7

L3

.13 mmol, 10 equiv.) was added to a solution (0.025 m) of 16

126 mg, 0.213 mmol, 1 equiv.) in DMSO (8.5 mL) and the mixture

C

-m GppCH

2

(

at 8–9 by addition of NaOH (0.1 m). After the reaction was com-

plete, the mixture was diluted with 10 volumes of water and the

product was isolated as an ammonium salt (3.5 mg, 2.6 μmol, 64%)

by semipreparative RP-HPLC with use of a linear gradient (0–

15%) of acetonitrile in ammonium acetate (pH 5.9, 0.05 m) over

was heated for 20 min in the microwave reactor with use of dynamic

power mode (Pmax = 5 W, Tmax = 45Ϯ1 °C). The excess of CDI

was then hydrolysed with water (65 μL, 3.61 mmol, 17 equiv.), and

6168

www.eurjoc.org

Eur. J. Org. Chem. 2015, 6153–6169

Amino- and Carboxy-Functionalised mRNA Cap Analogues

b

6

(

1

0 min (R

–): calcd. for C43

271.33097.

t

= 75.9 min). RP-HPLC: R

t

= 13.6 min. HRMS ESI-

H. Balci, Y. Ishitsuka, C. Buranachai, T. Ha, Annu. Rev. Bio-

chem. 2008, 77, 51–76; e) S. Preus, L. M. Wilhelmsson, Chem-

BioChem 2012, 13, 1990–2001.

–

_[_M_–_H_]– 1271.3364; found

H

66

N

14

O

23

P

3

S

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Received: May 25, 2015

Published Online: July 24, 2015

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Article Doi

mRNA Cap Modification through Carbamate Chemistry: Synthesis of Amino- and Carboxy-Functionalised Cap Analogues Suitable for Labelling and Bioconjugation

DOI: 10.1002/ejoc.201500672

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