- 1 -

Synthesis of protected 5’-aminouridine for modification of solid-support in synthesis of modified siRNA

A Thesis presented by

Swapna Suresh

to

The Department of Chemistry and Chemical Biology

In partial fulfillment of the requirements of the degree of the Masters of Science in the field of Chemistry

Northeastern University Boston, Massachusetts

- 2 -

Synthesis of protected 5’-aminouridine for modification of solid-support in synthesis of modified siRNA

by

Swapna Suresh

Abstract of Thesis

Submitted in partial fulfillment of the requirements for the degree of the Masters of Science in Chemistry

in the Graduate School of Arts and Sciences of Northeastern University, June 2008

- 3 -

ABSTRACT

My research was focused on the multi-step synthesis of solid support bearing 5’-

aminouridine for automated synthesis of siRNAs. The project started with the preparation

of 5’-azidouridine using either a Mitsonobu reaction with hydrazoic acid or a reaction of

uridine with lithium azide, triphenylphosphine and carbon tetrabromide. Azide was

hydrogenated to form 5'-aminouridine and further treated with para-methoxytrityl chloride

to protect the amino group. The generated 5'-amino-5’-N-methoxytrityluridine was then

treated with a solution of ortho-chlorobenzoyl chloride to protect the 2' hydroxyl group,

followed by succinic anhydride in the presence of 4-dimethylaminopyridine (DMAP) to

form a mixture of protected 2’ and 3' succinates. All new compounds were analyzed and

characterized using NMR techniques. The protected amino uridine succinate was then

loaded onto solid support – long chain aminoalkyl controlled pore glass (LCAA-CPG) to

be used in solid phase synthesis of amide modified siRNA.

The succinate moiety was coupled to the free amino groups on the CPG using N,N’-

dicyclohexylcarbodiimide (DCC ) N-hydroxybenzotriazole (HOBT ) to generate a stable

amide bond between the ribonucleoside and the CPG. The loading of the protected 5’-

aminouridine on CPG was quantified by following acid catalyzed detritylation at 478 nm

using a UV-VIS spectrometer.

The solid phase support will be used for preparation of amide modified siRNAs. We

propose that these non-ionic mimics of the phosphate backbone will confer high nuclease

resistance to siRNAs. Further, the backbone of the modified siRNAs will have an overall

decreased negative charge, which may help crossing the cellular membrane. We

anticipate that these properties may improve the biodistribution and pharmacokinetics of

modified siRNAs.

- 4 -

ACKNOWLEDGEMENTS

I would like to thank my research advisor Prof. Eriks Rozners for his guidance and support during

the course of my M.S thesis. He trained me on many lab techniques and gave me the confidence

and skills to solve many problems pertaining to my project. I would like to acknowledge Dr. Siji

Thomas and Dr. Martin Kullberg for their constant source of help and advice at each progressive

stage of my project.

I would also like to thank the members of my thesis committee, Prof. Eriks Rozners, Prof.

Graham Jones and Prof. David Forsyth for their patience in evaluating my thesis and providing

valuable advice to improve it.

Lastly, I would like to thank my family and friends for their support and encouragement throughout

these two years.

- - 5 - Table of Contents

Page Abstract 3

Acknowledgements 4

Table of contents 5

Glossary of Abbreviation 6

List of Figures 8

Chapter

1. Introduction

1.0- RNA interference 10

1.1- General Scheme of Solid Phase oligonucleotide Synthesis 13

1.2- Internucleoside Amide modified oligoribonucleotide 19

1.3- Solid support synthesis of MMT protected nucleosides 21

2. Results and Discussion

2.0- Objective 27

2.1- Reactions required for multi- step synthesis of solid support synthesis 28

2.2- Applications of the protected 5'-amino-3'-succinate solid support 36

3. Experimental Procedures

3.0- General Information 38

3.1- Experimental Procedures 39

3.2- 1HNMR and

13C NMR spectra 48

4. References 51

- 6 -

GLOSSARY OF ABBREIVIATIONS

ABREVIATION NAME

DNA Deoxynucleic Acid

RNA Ribonucleic Acid

siRNA short interfering RNAs

RISC RNA induced silencing complex

LNA Locked Nucleic Acid

DMTCl 4,4'-dimethoxytrityl chloride

MMTCl Monomethoxytrityl chloride

DMAP N,N-dimethylaminopyridine

LCAA-CPG Long chain alkyl amine- controlled pore glass

CF3COOH Trifluroacetic acid

CH2Cl2 Methylene chloride

DMF Dimethylformamide

DCC N,N'-dicyclohexylcarbodiimide

DNG Deoxynucleic guanidine

RNG Ribonucleic guanidine

DEAD Diethylazodicarboxylate

PPh3 Triphenylphosphine

CBr4 Carbon tetrabromide

LiN3 Lithium azide

CH3OH Methanol

HOBt N-Hydroxybenzotriazole

(CH3)3SiCl Trimethylsilyl chloride

CCl3COOH Trichloroacetic acid

MeCN Acetonitrile

H2SO4 Sulfuric acid

- 7 -

Na2SO4 Sodium sulfate

MgSO4 Magnesium sulfate

HN3 Hydrazoic acid

NaN3 Sodium azide

LiCl Lithium chloride

NH4OH Ammonium hydroxide

TEA Triethylamine

- 8 -

LIST OF FIGURES

Fig. No.

1. Diagrammatic Representation of RNA interference 10

2. Chemically modified RNAs 12

3. Phosphitylation of 3'- positions and coupling reaction 13

4. Scheme illustrating the Solid Phase synthesis of Oligoribonucleotides 16

5. Scheme representing the solid support synthesis using DCC and DMAP 18

6. Amide Modified Oligoribonucleotides 20

7. Solid support bearing 5’-monomethoxytritylamine protected 3'-O-succinate 21

8. Structure of DNG linkage 22

9. Synthesis of solid support bearing MMT protected 5'-aminodeoxyadenosine

and thymidine 23

10. Scheme representing the coupling of protected 5'-amino deoxyribonucleoside

3'-O-succinate and 3',5'-diamino monomer 24

11. Scheme representing the synthesis of solid support bearing

protected 5'-amino ribonucleoside 3'-O-succinate 25

12. Schematic Representation of the coupling of the protected 5'-amino

ribonucleoside 3'- succinate and 3', 5'-diamino monomer 26

13. Mitsonobu reaction of Uridine 28

14. Appel reaction of Uridine 29

15. Staudinger reaction of 5'-azido-5'-deoxyuridine 30

16. Monomethoxytritylation of the 5'-amino-5'-deoxyuridine 31

17. Benzoylation reaction of 5'-deoxy- 5'-N-monomethoxytritylaminouridine 31

18. Equilibrium reaction between the 3'-benzoate and the 2'-benzoate 32

19. Succinylation reactions of the 2'-benzoate and the 3'-benzoate 33

20. Solid support synthesis of the LCAA-CPG bearing

5'-N-monomethoxytritylamino protected amino uridine 35

- 9 -

21. Scheme illustrating single cycle of solid phase

oligoribonucleotide synthesis 37

-10 -

CHAPTER 1- INTRODUCTION

1.0- RNA Interference

The RNA interference is a gene control mechanism wherein the RNase II enzyme called the

Dicer cuts the double stranded RNA (dsRNA) into 21-22 base pairs long RNA duplexes called

small interfering RNAs (siRNAs). These siRNAs are unwound into the antisense and the sense

strand and the antisense strand (complementary to the target mRNA strand) is incorporated into

the RNA induced silencing complex called the RISC complex. The antisense strand binds to

complementary target mRNA strand and regulates the translational inhibition by degradation of

the complementary mRNA strand.1,2 Since the central dogma of biology states that DNA transfers

its genomic information to RNA which further translates this information into proteins, the RNA

interference prevents this second stage of information transfer by blocking the translation.3

Fig-1- Diagrammatic Representation of RNA interference3

- 11 -

The RNA interference is a natural pathway for silencing gene expression, which can be

harnessed to create innovative and potent drugs.2 Drugs based on the concept of RNA

interference will be applicable to large classes of proteins because there will be siRNAs for every

gene/ mRNA.2 Once the disease candidate is identified, the siRNAs will be developed using

bioinformatic concepts wherein the antisense strand will be complimentary to the target mRNA.2 4

Thus, the above mentioned properties will make the siRNAs applicable as RNAi drug candidates

to large classes of molecular targets.3

Role of Chemical Modification

Structurally, the siRNAs consist of two turns of a nucleic acid double helix, which makes them

large in size.2 The presence of the internucleoside phosphodiester backbone with approximately

40 anionic charges makes the siRNAs polar and hydrophilic, which will disable the crossing of the

lipophilic cellular membrane.2 Moreover, unmodified siRNAs will be easily degraded by

nucleases.2 Therefore, the exogenous siRNAs should be chemically modified to increase their

cellular uptake, potency, biodistribution and pharmacokinetics and minimize toxicity in the human

cells.2 In short, these siRNAs analogs should be chemically modified to make them more “drug

like”.4

Modifications of Ribose

Since the RISC recognizes the A-type geometry of the RNA helices, chemically modified siRNAs

should ideally adopt the A-type geometry of the RNA helix.5 Chemical modifications should be

well tolerated into the RNA helix and they should increase nuclease resistance as well as

increase the RNAi activity relative to the native siRNAs.5

There have been several chemically modified RNA analogs designed to increase the nuclease

resistance of the antisense strand.3 Since the RISC recognizes the overall shape of the A-type

helix of the modified siRNAs, some of these modifications such as the 2’-OMe,6-9 2’-F,8-12 locked

- 12 -

nucleic acids (LNA)11,12 and 4‘-thio RNA13 that reinforced the A- type geometry of the RNA have

shown promising results. However, 2'-deoxy-2'-flurouridine modification did not increase the RNAi

activity of the siRNAs.11 Modified siRNAs with boranophosphate13 and phosphorothioate10

internucleoside linkages were well tolerated in siRNAs and had increased RNAi activity.

The advances in nucleic acid chemistry have made the synthesis of modified oligoribonucleotides

from the 3' to the 5'- termini possible. The method of synthesizing these modified

oligoribonucleotides involves an automated solid phase procedure, which requires the “universal

solid support”. The universal solid support is solid support with an acid- labile linkage that is easily

cleavable after the completion of the synthesis of the modified oligoribonucleotides. Details of the

automated synthesis are discussed ahead.

O

OR

Base

O

OHO

Base

PO

-S O

O

OH

Base

O

OHO

Base

PO

H3B-O

O

OH

Base

S

OH

BaseO

F

BaseO

O

Base

Boranophosphates Phosphorothioates

2' -OR RNA

4'- thio RNA2'- F RNALocked NucleicAcids

Base

O

RO

O

P

OO

O

1'2'3'

4'

5'

R=CH2CH2OCH3

R=CH2(CH2)2CH3

R=CH2CH2F

R=CH2CF3

R=CH2CHCH2

R=CH2CCH

R=CH2CH2OCH2C6H5

N

N

R= H2CH2C

Fig- 2- Chemically modified RNAs

- 13 -

1.1- General Scheme of the Solid Phase oligoribonucleotide synthesis

Modified oligoribonucleotides are synthesized using automated solid- phase procedures. These

procedures involve phosphoramidite chemistry.14,15 Phosphoroamidites 2 are important building

blocks for the synthesis of oligoribonucleotides. The 3'-OH group is phosphitylated to generate

the 3'-O phosphoramidite, which is then coupled with the 5'- OH of another nucleoside 3 bound to

the solid support to generate the oligoribonucleotide dimer 4.14,15 (Fig- 3). The coupling reaction

increases the oligonucleotide chain to the desired length. 14,15

B1

O

OOHH

ODMT

TBS

2-Cyanoethyl N, N, N', N'-tetraisopropylphosphorodiamidite

CH3CN, N,N-disopropylamine-

tetrazolide, rt

B1

O

OOH

ODMT

TBSPNiPr

iPrO

(CH2)2CN

Phosphoramidite building block

B2

O

OOH

HO

TBSR2

Tetrazole

B1

O

OOH

ODMT

TBSPO

NC(H2C)2 B2

O

OOH

O

TBSR2

R2 = solid support

(1)

(2)

(3)

(4)

TBS= Si

Fig- 3 – Phosphitylation of 3'- positions and coupling reaction

Principles of solid phase oligoribonucleotide synthesis

The solid- phase synthesis of oligoribonucleotide (Fig 4) proceeds in the 3' to 5' direction in the

chain assembly. Thus, the solid phase strategy for the synthesis of such oligoribonucleotides

requires the combination of compatible protecting groups for the 2' and 5'–hydroxyl groups

wherein the 5'-terminal protecting groups must be selectively removed in

- 14 -

every cycle of the ribonucleotide addition and the 2'-protecting groups must remain intact

throughout such addition. The 2’- protecting group must be removed only in the end of the

oligonucleotide synthesis without cleavage of the internucleotide linkage. The protecting groups

are required because they prevent many possible side reactions from happening. The 5'- OH

group is temporarily protected during oligonucleotide synthesis to prevent self polymerization of

the monomers being added to each other.14,15

The 2' and 5'-protected ribonucleoside is reacted with succinic anhydride in the presence of a

nucleophilic catalyst, N, N,-dimethylaminopyridine (DMAP) to form the protected 3'-O-

succinate.14,15

The long chain alkyl amine (LCAA-CPG) is a widely used commercially available silica- based

support. The silica gel is treated with (3- aminopropyl) triethoxysilane to yield amino propyl- silica

which reacts with the carboxyl group of the protected 3'- succinate through a classical N,N'-

dicyclohexylcarbodiimide activation to form the succinyl linker.

Prior to the addition of each monomer, 5'-protecting group 5 bound to the solid support is

removed by a strong acid, 3% CF3COOH in CH2Cl2. The protecting group of choice is either 4, 4'-

dimethoxytrityl group (DMT) or monomethoxytrityl group (MMT). The cleavage of the DMT group

or the MMT group produces an orange color, which is extremely useful in monitoring the progress

of the reaction.14,15

After the removal of the 5'-dimethoxytrityl group, the ribonucleoside 6 is coupled with the 3'-

phosphoramidite building block 7 using tetrazole as the catalyst. The tetrazole protonates the N,

N-diisopropyl phosphoramidite and converts the diisopropylamino group into a good leaving

group.14,15

The nucleotide addition is completed by a “capping reaction” wherein the unreacted 5'-OH group

is acetylated in the presence of 1-methylimidazole and acetic anhydride followed by oxidation by

iodine to convert the phosphite triester to phosphate triester 9. The synthesis cycle is repeated

until the desired oligoribonucleotide chain is achieved.14,15

- 15 -

Subsequently, the fully protected support bound RNA is deprotected in a stepwise fashion. First,

treatment with aqueous ammonia cleaves the linkage to the solid phase and removes the

nucleobase and phosphate protecting groups. Then the 2'-O-protecting groups are removed. The

oligoribonucleotide prepared is purified using anion exchange High Performance Liquid

Chromatography.14,15

- 16 -

Base

O

OO

DMTO

O

O

HN

Step 1-CCl3COOH in CH2Cl2

Base

O

OO

HO

O

O

HN

TBSTBS

Base

O

OO

DMTO

P TBS

NiPr2

O

NC

Base

O

OO

O

O

O

HN

TBS

PO

Base

O

ORO

DMTO

CN

Step-2Condensation

Step 3 Oxidation

Iodine/water

Base

O

OO

O

O

O

HN

TBS

PO

Base

O

ORO

DMTO

CNO

(5)(6)

(7)

(8)

(9)

TetrazoleTBS= Si

Base= A, U , G, C

Fig- 4- Scheme illustrating the Solid Phase synthesis of Oligoribonucleotides

- 17 -

General procedure for the preparation of the solid support

The long chain alkyl amine (LCAA-CPG) is widely used commercially available silica- based solid

support.16 In a typical procedure, the solid support reacts with the protected 3'- succinate a DCC

and DMAP to form the universal succinyl linker. The solvents commonly used are CH2Cl2 and

DMF.16 CH2Cl2 is a good swelling agent for the support and the DMF increases the solubility of

the reactive species but excess of it could slow the rate of activation and hence minimal amounts

are added.16

DCC 10 is the activating species used to convert the carboxylic acid group of the succinate 11

into the O-acyl isourea intermediate 12.16 The carboxylic acid group of a second protected

ribonucleoside succinate donates its proton to the nitrogen atoms of the intermediate 13 and the

carboxylate ion generated attacks the carbonyl carbon of the acyl group to form the succinic

anhydride moiety 14.(Fig 5)16 The catalyst DMAP is added to 14 and the reaction forms a

succinylpyridinium ion 15 and a succinate ion 16. The amino group of the solid support LCAA-

CPG attacks the succinyl group of 15 and a stable amide bond between the protected

ribonucleoside 3'- succinate in 18 is formed. The general method of solid support synthesis

utilizes two equivalents of the 3'- succinate and 1 equivalent DCC and DMAP as a catalyst.

However after the addition of DMAP, only one equivalent of the succinate is coupled to the solid

support. The second equivalent of the ribonucleoside succinate is lost in the reactions.16

We had adopted a similar solid support synthetic strategy as stated above to prepare modified

oligoribonucleotides namely the amide modified oligoribonucleotides. The modified

ribonucleoside succinate was prepared and loaded onto the LCAA-CPG using peptide coupling

reactions. The solid support so prepared was used in the solid phase synthesis of amide modified

oligoribonucleotides. The amide modified oligoribonucleotides were incorporated into the

antisense strand of siRNA and the RNAi activity was studied.

-18 -

Base

O

OO

DMTO

TBSO

N

C

N +

O

HO

Base

O

OO

DMTO

TBSO

O

O

C

HN

NProtected-3'-succinate

DCC

H+Protonation

Base

O

OO

DMTO

TBSO

O

O

C

HN

NH+

Base= A, U , C, G

Base

O

OO

DMTO

TBSO

O

O 2

(10)

(11)

(12)

(13)(14)

(11)

DMAPN

N

Base

O

OO

DMTO

TBSO

O

N+

+

Base

O

OO

DMTO

TBSO

O

-O

N

NH2

(15)(16)

(17)

LCAA-CPG

Base

O

OO

DMTO

TBSO

O

NH

(18)

TBS= Si

Fig- 5- Scheme representing the solid support synthesis using DCC and DMAP

-19 -

1.2- Internucleoside Amide modified oligoribonucleotides

One of the main requirement of the RNA interference is that the antisense strand should be

nuclease resistant as well as it should show high binding affinity towards the target

complimentary mRNA strand. However, the presence of the negatively charged phosphodiester

linkages in the native antisense strand increases the electrostatic repulsion in the RNA-RNA

duplex and reduces the stability of the duplex.17

Ribonucleoside dimers with neutral and non-ionic dephospho linkages such as the thioformacetal

(3'- SCH2O-5')17, sulfide linkages, (3'-CH2CH2S-5')18, dimethylsulfone linkages, (3'-CH2SO2CH2-

5')19 , formacetal, (3'- SCH2O-5')18 were synthesized and incorporated in short DNA

fragments.16,17 Of these, the sulfide and the thioformacetal linkages destabilized the DNA-RNA

duplexes and the modified oligoribonucleotide analogs with dimethylsulfone linkages were not

able to hybridize well with oligodeoxynucleotides. The formacetal modification somewhat

decreased the stability of the DNA-RNA duplex.19

The synthesis and the properties of amide linked oligoribonucleotide analogs have been studied

extensively. Rozners et al. experimentally revealed that the hybridization of

oligodeoxyribonucleotide analogs with isomeric amide modifications amide linkages (3'-CH2-CO-

NH-5' (AM1 analogs) and 3'-CH2-NH-CO-5' (AM2 analogs), (Fig- 6) 21,22 with complimentary RNA

strand did not decrease the stability of the DNA-RNA duplex.20 The above modifications were

incorporated into ribonucleotide analogs that were made to hybridize with the complimentary

oligoribonucleotides to form the RNA-RNA duplex.20 UV melting experiments showed that such

isomeric amide modifications were well tolerated into the RNA-RNA duplexes.20,21 Furthermore,

the AM2 modification increased the stability of the duplex by 2 ºC.21,22

We therefore propose that the modified siRNAs having internucleoside amide linkages may have

better properties for therapeutic applications than the native siRNAs because these hydrophobic

- 20 -

non-ionic mimics of the phosphate backbone will not only show high stability towards nuclease

degradation, their backbone will have an overall decreased negative charge, which will decrease

the overall electrostatic repulsion of the duplex and help in crossing the lipophilic cellular

membrane. We anticipate that these properties will improve the biodistribution and

pharmacokinetics of amide modified siRNAs.

O

OH

Ura

O

HN

O

OHO

Ura

O

PO O

-O

PO

-O

O

OH

Ura

HN

C

O

OHO

Ura

O

PO O

-O

PO

-O

O

InternucleosideAmide (AM1) Linkage

3' 3'

5'5'

InternucleosideAmide (AM2) linkage

Fig- 6- Amide Modified Oligoribonucleotides

The internucleoside amide linkages (AM1 and AM2) have other advantages as well. 5,21,22

• Amides have well defined hydrogen donor and acceptor sites, which enable the modified

siRNAs to interact favorably with water and other biomolecules.

• The amide linkages in modified helices adopt a trans conformation, that is well

accommodated in A-type helices easily. Since RISC recognizes the overall feature of the

A-type RNA helices, these properties will make them as good internucleoside linkages in

RNA double helices.

• The amides can be synthesized using a relatively simplified methodology involving

peptide coupling protocols.

- 21 -

The RNA oligamides were previously synthesized by incorporation of amide linked uridine dimers

into the RNA structure.21, 24 However this method was laborious because it involved the synthesis

of large number of such dimers to construct all sorts of internucleoside amide linkages.20

Therefore we propose the automated solid support synthesis of amide linked oligoribonucleotides

for the preparation of amide modified siRNAs. The oligonucleotide synthesis will involve the direct

coupling of ribonucleoside monomers to form amide linkages on the solid support. These

coupling reactions will require the presence of the 3'-terminal building block which is attached to

the solid support with a succinyl linkage that is easily cleavable after the completion of the

synthesis of the amide modified oligoribonucleotides.

1.3- Solid support synthesis of MMT protected nucleosides

The solid support best suited for the synthesis of this amide linked oligoribonucleotides dimer is

proposed to be 5'-deoxy-5'-N-(4-methoxytritylamino)-2'-O-(ortho-chlorobenzoyl)-3'-O-succinyl-

uridine LCAA-CPG 19 unit or 5'-deoxy-5'-N-(4-methoxytritylamino)-3'-O-(ortho-chlorobenzoyl)-2'-

O-succinyl 20 ( Fig- 7)

U

O

OO

MMTHN

O Cl

O

O

NH

(19)

U

O

OO

O

MMTHN

O

O

HNCl

or

(20)

Fig 7- Solid support bearing 5’-monomethoxytritylamine protected 3'-O-succinate

One of the earliest solid supports bearing a 5'-N-monomethoxytritylaminouridine was synthesized

by Bruice et al, to prepare oligodeoxynucleotides with positively charged guanidinium linkages 21

(or the DNG oligomers).(Fig- 8) 25 These DNG oligomers formed a duplex with DNA oligomers 25.

- 22 -

The replacement of negatively charged phosphodiester bonds with positively charged

guanidinium linkages in DNG oligomers increased the DNG-DNA duplex stability due to

electrostatic attraction between the positively charged guanidinium linkages and the negatively

charged phosphodiester bonds. 25, 27, 28

O

HNH

HN

Base

H2N+

HN

Base= A and T

(21)

Fig- 8- Structure of DNG linkage

The building blocks for such DNG oligonucleotides were monomethoxytrityl (MMT) protected 5'-

amino-2'-deoxyadenosine and thymidine that were synthesized from the 2'-deoxyadenosine 22

and 2'-deoxythymidine 23, respectively (Fig- 10). 25 Initially 22 was modified into the dibenzoyl-5'-

O-mesyl derivative 24 by N-monobenzoylation of adenosine and the mesylation of the 5'-OH

group.25, Similarly 23 was converted to 25 by mesylation of the 5'-OH. 25, 27 Treatment with LiN3

converted 24 and 25 to the 5'-azido derivative which was followed by hydrogenation to the 5'-

amino derivative. 25 The 5'-amino group was selectively protected by the 4-methoxytrityl chloride

to give MMT protected 5'-amino-3'-OH monomers, 28 and 29. 25, Succination generated MMT

protected 5'-amino-3'-succinate deoxynucleosides 30 and 31, 25 which were then loaded onto the

long chain alkyl amine- controlled pore glass (LCAA- CPG) via the succinyl linkage.25 The

nucleoside loading was 36 µmol/g determined spectrophotometrically by the amount of MMT

cation released.(Fig 9) 28

- 23 -

O

HOH

HO

Base

O

HOH

MsO

Base

(22) Base= A(23) Base= T

(24) Base= ABz

(25) Base= T

O

HOH

N3

Base

(26) Base= ABz

(27) Base= T

ba

O

HOH

MMTHN

Base

(28) Base= ABz

(29) Base= T

c, d

eO

HO

MMTHN

Base

(30) Base= ABz

(31) Base= T

O O

HO

f,gO

HO

MMTHN

Base

O O

HN

(32) Base= ABz

(33) Base= T

Reagents and Conditions- a) for 1-3 (i) CH3SO2Cl, 1.0 eq, pyridine, 0ºC- rt , 9 hrs

(ii) TMSCl, 2.5 eq, 0ºC, (iii)- BzCl, 5 equiv, 0ºC, 3 hrs; for 2,4- CH3SO2Cl 1.0 equiv,

pyridine, 0ºC-rt, 9 hours; (b)- LiN3, 10.0 eq, DMF;(c)- 10% Pd/C , H2, EtOH, 4hours;(d)- pyridine, MMTCl, 1.0 eq; (e) succinic anhydride, 0.95 equiv, DMAP, pyridine,

12 hrs; (f) 4-nitrophenol, DCC, pyridine, 1,4- dioxane; (g) CPG, TEA, DMF, 12 hours

Fig- 9- Synthesis of solid support bearing MMT protected 5'-aminodeoxyadenosine and

thymidine 25

The thiourea in 3', 5'-diamino monomer was converted to carbodiimide group using HgCl2 and

triethylamine.25 The solid phase coupling of 3'-succinate 32 and 33 with the diamino monomer 34

formed the DNG oligomer 35 (Fig- 10)25

- 24 -

O

NH

HO

Base

H2N+

HNBase

O

NH

H2N+

HN

O

O

Base

4

Base= 5'-ATATTA-3'

O

O

MMTHN

Base

O O

HN

Base= ABz

Base= TO

O

NH

O

NH

MMTHN

Base

S

NH Fmoc

Solid phase synthesis

Protected 3'- succinate -1

3',5'-diaminomonomer

DNG oligomer(32) and(33)

(34)

(35)

Fig- 10- Scheme representing the coupling of protected 5'-amino deoxyribonucleoside 3'-

succinate and 3',5'-diamino monomer t

The positively charged guanidinium linkages have been used to replace the negatively charged

phosphodiester linkages of RNA to form polycationic ribonucleic guanidine (RNG).27,29 The

guanidinium linkages are nuclease resistant and stable to intermolecular nucleophilic attack by

the ribose C2'-OH. Bruice et al. used 5'-N-monomethoxytritylaminoprotected uridine for the solid

phase synthesis of the RNG oligomers. 27, 29

The 5'- monomethoxytritylamino derivative 36 was selectively silylated at the 2'-OH position by

tert-butyldimethylsilyl chloride (TBSCl) in the presence of silver nitrate and pyridine to form 37. 27,

29 Succination of 37 in the presence of DMAP as the nucleophilic catalyst gave the 3'-terminal

succinate 38. A solution of 3'-O-succinate 38 and 1-ethyl-3-[3-(dimethylamino) propyl]

carbodiimide hydrochloride was prepared and loaded to the aminopropyl controlled pore glass.26

The loading of 5'-monomethoxytritylamino protected 3’-O-succinate on the LCAA- CPG 39 was

determined to be 23.5 µmol/g. 27, 29

- 25 -

O

OO

MMTHN

Base

O

TBS

O

OH

O

OO

MMTHN

Base

H

TBS

O

OHO

MMTHN

Base

H

TBSCl

AgNO3Pyridine

DMAP

Succinicanhydrideroom temp.

DMFO

OO

MMTHN

Base

O

TBS

O

NH

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,room temperature

(36)(37)

(38)

(39)

NH2

Base= Uracil

SiTBS=

Fig- 11- Scheme representing the synthesis of solid support bearing protected 5'-amino

ribonucleoside 3'- succinate

The protected 5'-amino ribonucleoside 3'- succinate 39 so generated was coupled with 3', 5’-

diamino monomer 40 via solid phase synthesis (Fig- 12) to form polycationic ribonucleic

guanidine (RNG) oligomers.27, 28 The RNG was designed to form a duplex with a DNA strand

through stabilizing electrostatic attractive forces between the positively charged guanidinium

linkages in the RNG oligomers and the negatively charged phosphodiester linkages of the DNA

strand. 27, 28

- 26 -

O

NH

HO

Base

H2N+

HNBase

O

NH

H2N+

HN

O

O

Base

4

Base= 5'-ATATTA-3'

O

OTBSO

MMTHN

Base

O O

HN

Base= ABz

Base= TO

O

NH

O

NH

MMTHN

Base

S

NH Fmoc

Solid phase synthesis

Protected 3'- succinate -1

(40)3',5'-diaminomonomer

DNG oligomer

OTBS

OTBS

OTBS

OTBS

(39) (41)

Fig- 12- Schematic Representation of the solid phase synthesis of the protected 5'-amino

ribonucleoside 3'- succinate and 3', 5'-diamino monomer.

My M.S. project was focused on preparing a solid support that was very similar to that of Bruice et

al’s. However, unlike the solid supports shown in 39 that used the TBS group as the 2'-OH group,

I had used the 2-chlorobenzoyl chloride to block the 2'-OH group of the 5’-N-monomethoxytrityl

protected aminouridine. The newly formed benzoylated amino protected uridine was succinylated

and loaded onto the LCAA-CPG solid support using peptide coupling reactions. The support was

used by others in Prof. Rozner’s laboratory to prepare amide modified oligoribonucleotides.

- 27 -

CHAPTER 2 - RESULTS AND DISCUSSION

2.0- Objective

The goal of this research project is to develop multi step synthesis of solid support bearing 5’-

aminouridine for automated synthesis of siRNAs. The synthesis started with preparation of 5'-

azidouridine and reduction of the azido group to the amino functionality. The 5'-aminouridine was

tritylated to generate the 5'-monomethoxytritylated product. The trityl compound was selectively

benzoylated at the 2'-position and succinated to give the protected 3'-ribonucleoside succinate.

The 3'-terminal nucleoside was anchored via a succinyl linkage to the amino functionalized

controlled pore glass (CPG). The amino derivatized solid support was used by others in the lab to

prepare amide modified siRNAs.

28 -

2.1- Reactions required for the multi-step synthesis of the solid- support

i)- Preparation of 5’-azido-5’-deoxyuridine via Mitsonobu reaction

The synthesis of the 5'-azido-5'-deoxyuridine 42 via Mitsonobu reaction using hydrazoic acid in

the presence of diethylazodicarboxylate (DEAD) and triphenylphosphine was adopted from the

literature procedure (Figure 13).33 The triphenylphosphine was combined with DEAD to generate

the phosphonium intermediate that added to the 5’-OH of uridine to form 44. The hydrazoic acid

was the nitrogen nucleophile that attacked the phosphonium ion intermediate via an SN2

mechanism to generate 5'-azido-5'-deoxyuridine and triphenylphosphine oxide. The product was

obtained in 90% yield as thick white foam.

NH

O

ON

O

OHOH

HO

(42)

NH

O

ON

O

OHOH

OPh3P+

DEAD

PPh3

NH

O

ON

O

OHOH

N3

HN3

(43) (44)

Fig- 13- Mitsonobu reaction of Uridine

ii). Preparation of 5'-azido- 5'-deoxyuridine via Appel Reaction

An alternative way to prepare 5'-azido-5'-deoxyuridine was by the Appel reaction. The literature

procedure 34 to prepare the 5'-azido derivative 42 involved mixing uridine with LiN3 in DMF under

inert atmosphere (Fig 14). Hygroscopic CBr4 and triphenylphosphine were added to the reaction

mixture and the reaction was stirred overnight. The triphenylphosphine was activated by carbon

tetrabromide to form the phosphonium ion intermediate which added to the 5’-OH of uridine to

form 44. The LiN3 nucleophilically attacked the 44 to form the 5'-azido-5'-deoxyuridine. The 5'-

azido-5'-deoxyuridine was obtained in 87% yield as thick white foam. The reaction was adopted

because it avoided the use of toxic chemicals such as DEAD and HN3 that were used in the

Mitsonobu reaction. 33

- 29 -

PPh3

CBr4

NH

O

ON

O

OHOH

O

P+Ph Ph

Ph

(43)

NH

O

ON

O

OHOH

N3

(44)

NH

O

ON

O

OHOH

O

H

(42)

LiN3

Fig- 14- Appel reaction of Uridine

iii) Preparation of 5'-amino- 5'-deoxyuridine via Staudinger Reaction and Hydrogenation

reaction

5'-Azido-5'-deoxyuridine 44 was reduced to 5'-amino-5'-deoxyuridine 47 by Staudinger reaction

Figure 15).36 Triphenylphosphine reacted with the azide to generate the phosphazide 45, which

lost nitrogen to generate the iminophosphorane 46. Quenching the reaction with ammonium

hydroxide formed the 47and triphenylphosphine oxide. However the reduction by Staudinger

reaction did not proceed to completion. There were trace amounts of 44 present in the reaction

mixture. Thus, the yield of 95 % was not the true yield. Furthermore, triphenylphosphine oxide

was formed during the reaction which was never eliminated with ease. To circumvent the above

mentioned drawbacks, the 5'-azido-5'-deoxyuridine was hydrogenated in the presence of Pd/C in

dry methanol. The hydrogenation proceeded to completion to generate 5'-amino-5'-deoxyuridine

in 95% yield.36The reaction was simpler and did not generate any side- products.

- 30 -

NH

O

ON

O

OHOH

N3

NH

O

ON

O

OHOH

N

NN

PPh

PhPh

Phosphazide

NH

O

ON

O

OHOH

NPPhPh

Ph

Iminophosphorane

PPh3N2

NH

O

ON

O

OHOH

H2N

(47)

(44) (45)(46)

Fig- 15- Staudinger reaction of 5'-azido-5'-deoxyuridine

iv) Preparation of the 5'-deoxy- 5'-N-monomethoxytritylaminouridine

Compound 47 was mixed with MMTCl 48 in pyridine and the reaction was stirred overnight

(Figure 16).27 The MMT group selectively protected the 5'-amino function of uridine. However, the

MMTCl compound was very hygroscopic. Thus, reaction was stirred under inert atmosphere for a

few hours after which the nitrogen line was removed and the reaction was allowed to stir

overnight. 5'-deoxy-5'-N-monomethoxytritylaminouridine 49 was formed in 59% yield as light

brown foam.

- 31 -

NH

O

ON

O

OHOH

H2N+

Cl

OCH3

NH

O

ON

O

OHOH

HNH3CO

N

- HCl

(49)(48)

-(47)

Fig- 16- Monomethoxytritylation of the 5'-amino-5'-deoxyuridine

v) Benzoylation reaction

Compound 43 was converted to a mixture of 3'- and 2'-succinates 47 and 48 by a novel

procedure involving selective benzoylation of 49 at the 2'-OH by 2-chlorobenzoyl chloride at –78

0C and succination at the 3'-OH by succinic anhydride and DMAP. The benzoylation and the

succination reactions occurred sequentially in a single pot. The 2-chlorobenzoyl chloride was

chosen as the 2'- protecting group because its use was compatible with the 5'–protecting group,

i.e., the MMT group. It was mostly selective for the 2'-OH group. The selective benzoylation to

give 2'-O-orthochlorobenzoyl product 50 was kinetically driven. (Fig- 17)

NH

O

ON

O

OHOH

MMTHNO

Cl

NH

O

ON

O

OOH

MMTHN

O

Cl

CH2Cl2 -780C+

NH

O

ON

O

OHO

MMTHN

O

Cl

(50)- 95% (51)- 5%

Cl

(49)

Fig- 17- Benzoylation reaction of 5'-deoxy- 5'-N-monomethoxytritylaminouridine

The formation of the kinetically driven 2'-O-benzoate product has been explained by steric and

electronic effects. The electron withdrawing effects of the nitrogen and the ribose ring oxygen

made the anomeric carbon electron-deficient, which in turn exerted its electron withdrawing

- 32 -

inductive effect on the 2’-carbon. The electronic effects experienced by the 2’-carbon increased

the acidity of the 2'- OH and made it more reactive for attack by the 2-chlorobenzoyl chloride.

Furthermore, the bulky 5'-MMT group sterically hindered the 2-chlorobenzoyl chloride from

benzoylating the 3'-OH.

vi) Preparation of protected 3' succinate and protected 2'-succinate-

The formation of the 3'-O-orthochlorobenzoylated product was assumed to be thermodynamically

driven. The DMAP catalyzed the equilibrium between the 3'-benzoate with the kinetically favored

2'-benzoate. The mechanism of conversion is shown below. The lone pair of electrons on the 2'-

oxygen of 51 attacked the carbonyl carbon to form the five-membered intermediate 52. The

intermediate rearranged to generate the 2'-benzoate 50. Rearrangement of the intermediate to

the 2'- benzoate was possible by the 1,5- interaction in the ribose sugar. (Fig- 18). Here U is the

Uracil base.

O

OO

MMTHN U

O

O-O

MMTHN U

O

Cl

O

OO-

MMTHN

O

Cl

U

-O

Cl

(51) (52) (50)

Fig- 18- Equilibrium reaction between the 3'-benzoate and the 2'-benzoate

The equilibrium eventually shifted towards the formation of the thermodynamically more stable 3'-

benzoate. The reaction mixture presumably consisted of the 2'- benzoate 50 and the 3'-benzoate

51 in the ratio of (1:4) respectively. Succination of the reaction mixture resulted in

monomethoxytrityl protected 3'-O-succinate 53 and the 2'-O-succinate 54 in the same ratio.( Fig-

19)

- 33 -

O

OOH

MMTHN

O

Cl

U

O

OHO

MMTHN U

O

Cl

DMAP

O

O

OU

O

OO

O

MMTHN

O

OHOCl

(53)

+

U

O

OOO

ClO

O

HO

MMTHN

(52)

(1:4)

(50) (51)

(54)

U= Uracil

Fig- 19- Succinylation reactions of the 2'-benzoate and the 3'-benzoate

vi) Preparation of the solid support

The long- chain alkyl amine controlled pore glass; LCAA-CPG of 500- 1000 Å was used as the

solid support. The traditional procedure of the solid support synthesis required the presence of

DMAP as a coupling additive. However, our scheme of loading the 5'-monomethoxytritylamino

protected 3’-succinate on the LCAA- CPG, used N-hydroxybenzotriazole HOBt 52 as a coupling

additive.

Protected 5’-aminouridine succinates (the 3'-O-succinate and the 2'-O-succinate isomers) 53 and

54 were dissolved in dry CH2Cl2. N, N’-Dicyclohexylcarbodiimide (DCC) 10 and HOBt 56 were

added to succinates to give the activated HOBt ester 58 .The amino group of the solid support

- 34 -

nucleophilically attacked the carbonyl group and the HOBt was expelled. The solid support was

thus coupled to the monomethoxytrityl protected amino uridine by a stable amide bond 59 and 60

(Fig- 20).

The solid support synthesis involving HOBt required and consumed only 1 equivalent of

succinate. Our scheme of solid support synthesis proved advantageous over the traditional

procedure because it used only one equivalent of succinate to react with 1 equivalent of DCC and

DMAP. Whereas the traditional procedure required 2 equivalents of the ribonucleoside succinates

to react with 1 equivalent of DCC and DMAP but only one equivalent of succinate was able to

couple to the solid support. Thus, the general scheme resulted in the loss of the prepared

succinate. However, no loss of the succinate was observed in our scheme of solid support

synthesis. Therefore, the use of HOBt increases the efficiency of the solid support synthesis.

After the addition of DCC and HOBt, the mixture was filtered on the LCAA-CPG support and the

slurry was stirred for 24 hours. The support was filtered, washed with CH2Cl2, methanol and

acetonitrile. The unreacted amino groups was capped by treating the support with the mixture of

acetic anhydride/N-methylimidazole/2,6-lutidine/MeCN the support was washed with the mixture

of CH2Cl2 and methanol for 10 minutes and dried in vacuum.

The loading of the 5'-N-monomethoxytritylamino-3'-O-succinate and 2'-O-succinate on the solid

support was quantified by letting a small sample of the resin to react with 3% CCl3COOH in

CH2Cl2 and determining the monomethoxytrityl cation concentration by UV- VIS spectroscopy

(478 nm). This loading was 5'-monomethoxytritylamino protected 3’- succinate on the LCAA-

CPG was 46 µmole/g. This value was similar to typical nucleoside loadings used for the

automated synthesis of the oligoribonucleotides The above method of functionalization of the

solid support was compatible with the solid phase synthesis of amide modified oligoribonucleotide

from the 3' to the 5' direction.

- 35 -

NH

ON

O

OO

MMTNH

O

O Cl

N

C

N +

O

HO

NH

ON

O

OO

MMTNH

O

O Cl

O

O

C

HN

N

NH2NH

O

ON

O

OO

MMTNH

O

O Cl

O

HN

Dicyclohexylcarbodiimide Protected-3'-succinateand 2'-succinate (54)

solid support bearing monomethoxytritylaminoprotected 5'-amino-3'-succinate (59) and 2'-succinate (60)

N

N

N

OH

NH

O

ON

O

OO

MMTNH

O

O

O

O

C

HN

NH+

N

N

N

O-

NH

O

ON

O

OO

MMTHN

O

O

O

O

N

Cl

NN

Cl

(10)

(53)

(55)

(56)(57)(58)

(59)

O O

Fig- 20- Solid support synthesis of the LCAA-CPG bearing 5'-N-monomethoxytritylamino

protected amino uridine

- 36 -

2.2- Application of the protected 5'-amino-3'-succinate solid support.

The protected 5'-amino-3'-succinate solid support, 61 was used by Dr. Kullberg in Prof. Rozner’s

laboratory to couple with the monomer 2'-O-Acetyl-3'-carboxymethyl-5'-(monomethoxytrityl)

aminouridine triethylammonium salt 62 to form dimer 4. The dimer so formed had an amide bond

formed between the two ribonucleosides 64. The cycle of solid phase synthesis was repeated to

obtain oligoribonucleotide 3'-UUCUUUCUCGUAGAUGCCACU-5’, the antisense strand of a

siRNA that targets the cyclopilin B gene. The bold letters designate ribonucleotide units having

amide internucleoside linkages. . (Fig- 21)

- 37 -

U

O

OO

MMTHN

O Cl

O

O

NH

U

O

OO

H2N

O Cl

O

O

NH

B

OMMTNH

B

O

OAc

MMTNH

OU

O

OO

HN

O Cl

O

O

NH

Capping

CouplingReaction

B

O

OAc

MMTNH

OU

O

OO

HN

O Cl

O

O

NH

Detritylations

ProtectedOligonucleotide

NH3/ MeOH

Oligonucleotide

3% CCl3COOH

CH2Cl2

(59) and

(60) (61) (62)

(63)

(64)

(66)

O

OH

B= Uracil-1-ylB= C-N-Bz-cytosin-1-ylU= Uracil

(65)

Fig- 21- Scheme illustrating single cycle of solid phase oligoribonucleotide synthesis

- 38 -

CHAPTER 3- EXPERIMENTAL PROCEDURES

3.0-General Information

All reactions were carried out in dry glassware (dried in the oven at 145 0C overnight and sealed

with a rubber septum, cooled under nitrogen).

All syringes and needles were stored in the oven overnight prior to use and were allowed to cool

in the desiccators. Reactions were stirred with Teflon coated magnetic stir bar. Experiments were

monitored using Thin Layer Chromatography. All solvents were purchased from Aldrich. DMF,

THF and pyridine were dry distilled. 1H NMR and 13C NMR spectra were recorded on a 300 MHz

Varian Mercury instrument. Both 1H NMR and 13C NMR were presented in ppm downfield relative

to tetramethylsilane as an internal standard. The TLC analyses were carried out on general

purpose silica gel on polyester TLC plates and were visualized under UV light and by staining

with concentrated H2SO4.

Chromatographic separations were made using Silica Flash 40- 63 µm 60 Å purchased from

Silicycle.

- 39 -

3.1- Experimental Procedures

Mitsonobu Reaction to prepare 5’-azido- 5’-deoxyuridine

NH

O

ON

O

OHOH

HO

NH

O

ON

O

OHOH

N3

5'-azido-5'-deoxyuridine

HN3 PPh3

DEAD

Uridine

Preparation of hydrazoic acid. Sodium azide (13.51 g, 0.205 mmole) was dissolved in H2O (8

mL) and toluene (98 mL). The solution was maintained at 0 ºC. Concentrated H2SO4 (7 mL) was

slowly added to the solution of sodium azide. The reaction was maintained at or below 0 ºC. The

mixture was stirred for 30 minutes to an hour at which time Na2SO4 precipitated as a white solid.

The toluene layer containing hydrazoic acid was decanted and dried over MgSO4 and was used

immediately in the next step.

Preparation of 5’-azido-5’-deoxyuridine. Uridine (2.00 g, 8.1 mmole) was dissolved in dry

tetrahydrofuran under inert atmosphere. Hydrazoic acid in toluene (10 mL) was added drop wise

to the reaction and the mixture was cooled to 0 ºC. Triphenylphosphine (6.42 g, 24.3 mmole) was

added to the solution. (DEAD) (3.8 mL, 24.5 mmole) was added drop wise and the reaction was

allowed to warm up to room temperature. The reaction was stirred at room temperature. After 24

hours, the reaction was concentrated under reduced pressure. The resulting viscous liquid was

extracted with minimal amount of water and CH2Cl2. The resulting water layer containing the

azido compound was freeze dried and the solid residue obtained purified on a column

chromatography (8-10 % methanol in CH2Cl2) to afford the 5’-azido-5’-deoxyuridine.Yield: 1.998

g, 90% yield. TLC Rf = 0.36 CH3OH /CH2Cl2 (1:9). NMR data was comparable to that given in the

literature procedure.33

- 40 -

Alternative strategy to prepare 5’- azido-5’-deoxyuridine via Appel reaction

LiCl NaN3+ LiN3 + NaClMeOH

Preparation of lithium azide. Sodium azide (3.37 g, 51.00 mmole) was mixed with lithium

chloride (1.987 g, 46.8 mmole ) in dry methanol (5 mL) under inert atmosphere. The reaction was

stirred under inert atmosphere at 60ºC for 24 hours. The thick white paste formed was stored in

the freezer for 3 hours to precipitate the sodium chloride as a white residue. The reaction mixture

was then filtered and washed with methanol. The filtrate was concentrated under vacuum to

generate lithium azide as a white solid.

LiN3, PPh3

CBr3, DMF

NH

O

ON

O

OHOH

HO

Uridine

NH

O

ON

O

OHOH

N3

5'-azido-5'-deoxyuridine

Preparation of 5’-azido-5’-deoxyuridine: Uridine (0.628 g, 2.57 mmole) was mixed with lithium

azide (0.500 g, 10.3 mmoles) in dry DMF (6 mL). The reaction mixture was concentrated in vacuo

till approximately half of the DMF was evaporated off. The reaction mixture was then stirred under

inert atmosphere and triphenylphosphine (0.979 g, 3.73 mmole) and carbon tetrabromide (1.280

g, 3.86 mmole) were added. The reaction mixture was stirred at room temperature for 24 hours

and quenched with methanol (2 mL). The reaction mixture was concentrated under vacuum and

purified by silica gel chromatography (6-8% methanol in CH2Cl2) to form 5’-azido-5’-deoxyuridine

Yield: 0.6072 g, 87.7%. TLC Rf = 0.33 CH3OH:CH2Cl2 (1:9). NMR data was comparable to that

given in the literature procedure.33

- 41 -

Preparation of 5’-amino-5’-deoxyuridine by Staudinger reaction

NH

O

ON

O

OHOH

N3

NH

O

ON

O

OHOH

H2NPPh3, NH3

Pyridine

5'-azido-5'-deoxyuridine 5'-amino-5'-deoxyuridine

5’-azido-5’-deoxyuridine (0.7646 g, 2.84 mmole) was coevaporated with pyridine (3 x 10mL). 5’-

Azido-5’-deoxyuridine was mixed with triphenylphosphine (1.489 g, 5.68 mmole) in pyridine (10

mL). The reaction mixture was stirred for 3-4 hours after which concentrated ammonium

hydroxide (4 mL) was added drop wise. The reaction mixture was stirred overnight. The solution

was concentrated in vacuum and water was added to precipitate out triphenylphosphine oxide as

white residue. Triphenylphosphine oxide was filtered off and the aqueous solution containing 5’-

amino-5’-deoxyuridine was extracted with benzene and diethyl ether or ethyl acetate to remove

the residual triphenylphosphine oxide. The aqueous layer was concentrated to dryness. Yield:

0.6592 g, 95%.TLC Rf = 0.09 CH3OH/CH2Cl2 (1:9). But the TLC of the reaction mixture revealed

the presence of the 5'-azido-5' deoxyuridine in the reaction mixture. So the reaction did not

proceed to completion.

- 42 -

Preparation of 5’- amino- 5’-deoxyuridine by palladium catalyzed hydrogenation.

Pd/ C H2

MeOH

NH

O

ON

O

OHOH

N3

5'-azido-5'-deoxyuridine

NH

O

ON

O

OHOH

H2N

5' amino-5'-deoxyuridine

5’-azido-5’-deoxyuridine (1.078, 4.00 mmole) was dissolved in dry methanol (10 mL) under inert

atmosphere. Palladium on carbon catalyst (0.219 g) was added to the solution. The reaction was

then stirred at room temperature under hydrogen atmosphere (balloons) for 1 day. Yield: 0.954 g,

98%. TLC Rf = 0.09 CH3OH/ CH2Cl2 (1:9).

NMR data was comparable to that given in the literature procedure.35

- 43 -

Preparation of 5’-N-methoxytritylamino-5'-deoxyuridine

NH

O

ON

O

OHOH

H2N +

Cl

OCH3

NH

O

ON

O

OHOH

HNH3CO

N

- HCl

5'-amino-5'-deoxyuridine 5'-deoxy-5'-N-methoxytritylaminouridineMonomethoxytritylchloride (MMTCl)

-

5’-amino-5’-deoxyuridine (0.5141 g, 2.11 mmole) was coevaporated with pyridine (3 x 10 mL) and

dissolved in pyridine (10 mL). MMTCl (1.958 g, 6.34 mmole) was added and the reaction mixture

was stirred under inert atmosphere overnight. The reaction was quenched using aqueous

ethanol. Excess pyridine was evaporated under vacuum. The viscous orange liquid was dissolved

in ethyl acetate and the organic layer was extracted with water, concentrated aqueous sodium

bicarbonate and concentrated aqueous sodium chloride. The organic layer was dried (Na2SO4),

concentrated and purified using column chromatography (5% methanol in CH2Cl2 containing

0.1% triethylamine) to form 5’-deoxy-5'-N-methoxytritylaminouridine. Yield: 0. 412 g, 59%.TLC Rf

= 0.5 CH3OH/ CH2Cl2 (1:9) 1H NMR data (CDCl3 ,300 MHz) δ: 2.3 ( dd, J = 18 Hz, 1H,) , 2.7 (d,

J = 12 Hz, 1H) , 3.75 (s, 3H), 3.9 ( t, J = 11.4, 1H) , 4.1 ( d, J = 28.5, 2H) , 5.5 ( d, 9Hz, 1H), 5.8 (

s, 1H) , 6.8 ( d, J = 27, 1H), 7.1- 7.4 (m, J = 96 Hz, 13 Hz). 13C NMR (CDCl3) δ 46.0, 60.4, 74.0,

80.0, 100.3, 141.13, 148.1, 153.7, 116.0- 140.

- 44 -

Preparation of 5'-deoxy-5'-N-(4-monomethoxytritylamino)- 2'-O- (orthochlorobenzoyl)-3'-O-

succinyluridine

NH

O

ON

O

OHOH

MMTNH

Cl

O

OCl

O

O

O

DMAP

Pyridine,

5'-deoxy-5'-N-monomethoxytritylaminouridine

NH

O

ON

O

OO

MMTNH

O O Cl

5'-N-methoxytritylamino-2'-O-mono(orthochlorobenzoyl)-3'-O-succinyluridine

O

HO

+

NH

O

ON

O

OO

MMTNH

O O

5'-N-methoxytritylamino-3'-O-mono(orthochlorobenzoyl)-2'-O-succinyluridine

O

OH

Cl

5’-deoxy-5’-N-methoxytritylaminouridine (0.5899 g, 1.14 mmole) was co evaporated with dry

pyridine (3 x 10 mL) and dissolved in dry CH2Cl2 (15 mL). Pyridine (1 mL) was added and the

reaction was cooled to –78 ºC (acetone-dry ice). A solution of 2-chlorobenzoyl chloride (0.16 ml,

1.25 mmole) in dry CH2Cl2 (2 mL) was added. The mixture was stirred at -78ºC for 30 minutes

after which succinic anhydride (0.1259 g, 1.25 mmole) and of DMAP (0.2096 g, 1.71 mmole)

were added simultaneously. The reaction was stirred for 24 hours at room temperature. The

reaction mixture was concentrated and the viscous residue was washed with water and

concentrated aqueous sodium bicarbonate. The organic layer was dried (Na2SO4,) and

concentrated. The residue (containing the two isomers of the protected ribonucleoside succinate)

was purified by silica gel column chromatography (6%- 8% methanol in CH2Cl2 containing 0.1%

triethylamine). Yield: 5'N-monomethoxytritylamino protected ribonucleoside 2'-O-succinate-

- 45 -

0.100g, 11.5%. TLC Rf = 0.69 CH3OH/ CH2Cl2 (1:9) 5 'N-monomethoxytritylamino protected

ribonucleoside 3'-O-succinate- 0.010g, 1.15%. TLC Rf = 0.45 CH3OH/ CH2Cl2 (1:9)

The succination in the presence of DMAP as a nucleophilic catalyst gave a mixture of isomers

namely: the 5'-N-monomethoxytritylamino protected 3'-O-succinate and the 2'-O-succinate in the

ratio of 1:4 respectively. Thus, the protected 2'-O-succinate is the dominant isomer.

1H NMR data (CDCl3, 300 MHz) - 2.3 (dd, J = 18 Hz, 1H,), 2.7 (d, J = 12 Hz, 1H) , 3.75 (s, 3H),

5.5 (s, 2H), 5.69 (d, J = 21 Hz, 1H), 6.0 (d, J = 6H, 1H), 6.8 (d, J = 9 Hz, 2H), 7.8 (d, J = 6 Hz, 1H)

- 46 -

Preparation of solid support bearing protected aminouridine

NH

O

ON

O

OO

MMTNH

O O Cl

5'-deoxy-5'-N-(4-monomethoxytritylamino)-2'-O- (orthochlorobenzoyl)-3'-O-succinyluridine

NH2NH

O

ON

O

OO

MMTNH

O O Cl

Solid support bearing protected aminouridine

HOBt, DCC

O

HO O

HN

Long chain aminoalkyl controlled pore glass (LCAA-CPG) (400 mg) was placed in a round

bottomed flask equipped with a glass frit filter side arm. Trimethylsilylchloride (2mL) and pyridine

(4 ml) were added and the reaction mixture was stirred for two hours at room temperature. The

reagents were removed through the filter side arm by applying positive pressure of nitrogen. The

solid support was washed with dry pyridine (2 mL), dry CH2Cl2 (2 x 2 mL), and dry methanol (2

mL). The solid support was treated with piperideine (1 mL) in DMF (4 mL) for 10 minutes at room

temperature. The support was filtered and washed with dry DMF (2 x 2 mL) and CH2Cl2 (3 x 2

mL).

Protected 5’-aminouridine succinate (0.083 g, 0.110 mmole) was co evaporated with dry pyridine

(3 x 5 mL) and dissolved in dry CH2Cl2 (4 mL). DCC (0.022 g, 0.11 mmole) and HOBt (0.0148 g,

0.11 mmole) were added and the reaction mixture was stirred for 30 minutes. The mixture was

filtered on the LCAA-CPG support and the slurry was stirred for 24 hours. The support was

filtered, washed with CH2Cl2 (3 x 2 mL), methanol (2 mL), Acetonitrile (2 x 2 mL). The unreacted

amino groups was capped by treating the support with the mixture of acetic anhydride/ N-

methylimidazole/2,6-lutidine/MeCN (1:1:1:7, 10 mL),the support was washed with the mixture of

CH2Cl2 and methanol (1:1, 5 mL ) for 10 minutes and dried in vacuum.

- 47 -

Detritylation reaction to prepare amino derivatized solid support

NH

O

ON

O

OO

MMTNH

O O Cl

Solid support bearingprotected aminouridine

3% CF3COOH

CH2Cl2

NH

O

ON

O

OO

H2N

O O Cl

O O

HOHO

5'-deoxy- 2'-O- (orthochlorobenzoyl)-3'-O-succinyl-5'aminouridine

The solid support (0.0069 g) was placed into a vial and 3% CF3COOH in CH2Cl2 (1 mL) was

added. The color of changed from transparent to yellowish-orange. The solution was transferred

to a volumetric 10 mL flask and diluted with 3% CF3COOH in CH2Cl2. The solution was

quantitated on a US-VIS spectrometer using the absorbance of monomethoxytrityl cation at 478

nm. The amount of the aminouridine loaded on the support was calculated as::

Loading µ mol/g = (Absorbance * Volume (dilution factor)) / (56.11 * mass support)

= (0.451 x 40) / (56.11 x 0.0069)

= (18.04/ 0.387) = 46.60 µ mol / g

The support loading was 46 µ mol / g

- 48 -

- 49 -

- 50 -

- 51 -

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