Enabling Multiple Conjugation to Oligonucleotides Using âClick Cycles

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Enabling multiple conjugation to oligonucleotides using “click cycles” Martina Jezowska-Herrera, Dmytro Honcharenko, Alice Ghidini, Roger Stromberg, and Malgorzata Honcharenko Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00380 • Publication Date (Web): 19 Oct 2016 Downloaded from http://pubs.acs.org on October 20, 2016

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Bioconjugate Chemistry

Enabling multiple conjugation to oligonucleotides using “click cycles”. Martina Jezowska1, Dmytro Honcharenko1, Alice Ghidini1, Roger Strömberg*1, Malgorzata Honcharenko*1 1

Department of Biosciences and Nutrition, Karolinska Institute, Novum, SE-14183 Huddinge, Sweden

* Corresponding authors: Malgorzata Honcharenko, Tel: +46-8-52481025; E-mail:

[email protected], and Roger Strömberg, Tel: +46-8-52481024; E-mail: [email protected] , Department of Biosciences and Nutrition, Karolinska Institute, Novum, SE-14183 Huddinge, Sweden

Abstract An efficient method for synthesis of multiply functionalized oligonucleotides (ONs) utilizing a novel H-phosphonate-alkyne based Linker for Multiple Functionalization (LMF) is developed. The strategy allows for conjugation of various active entities to oligonucleotide through the post-synthetic attachment of LMF at the 5’-terminus of ONs using Hphosphonate chemistry followed by conjugation of various entities via [3+2] copper(I) catalysed cycloaddition in a stepwise manner. Each cycle is composed of attachment of the LMF followed by click reaction with azido containing units. Sequential solid-phase synthesis of oligonucleotide conjugates containing three attached entities was performed using acetylated form of MIF peptide conjugated to azido linker, achieving high conversions at each unit addition. In addition, to show the versatility of the method, oligonucleotide conjugates with several different classes of compounds were synthesized. Each conjugate containing three different entities, whose structure and function varied (e.g., sugars, peptides, fluorescent labels, m3G-Caps).

Introduction Therapeutic oligonucleotides (ONs) provide opportunity for treating serious, life-threatening diseases with limited options using traditional small-molecule and antibody drugs. Side effects of antisense approaches are relatively limited and correlated with when high doses of

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therapeutic oligonucleotides are needed for efficacy. Nevertheless, numerous oligonucleotidebased therapeutics are being tested in clinical trials. Examples include use of a receptortargeted siRNA conjugate, strong effects on liver diseases using antisense with novel chemical modifications, anti-cancer effects with a miRNA, and treatment of a neurodegenerative disease via intrathecal administration of a SSO (Splice Switching Oligonucleotide).1 Preparation of successful oligonucleotide based therapeutics is a substantial challenge. Numerous modifications and conjugates have been reported in order to address the limitations connected with the use of unmodified oligonucleotides. The poor delivery, tissue selectivity and intracellular distribution can be improved, e.g., by the attachment of cellpenetrating peptides (CPPs)2-4, homing peptides that allow targeting specific cells or tissues57

, and nuclear localization signals (NLS) enabling more efficient transport to the cell

nucleus.8-12 In addition, fluorescent labelling or other tagging of oligonucleotides is essential for visualization of transport pathways and assessment of effects of conjugates. However, multiple functionalization of the ONs in order to combine the effect of various entities brings additional challenges. Attachment of several entities to oligonucleotides by use of click chemistry has been done by Carell and co-workers in preparation of DNA constructs with different fluorescent labels13. The method is generally limited to two cycloadditions per oligonucleotide due to that the authors uses protected-alkyne and alkyne moieties that are both connected to the oligonucleotide when the first click reaction is performed. After the first click attachment to the alkyne group, deprotection of the protected-alkyne moiety is needed, followed by a second conjugation reaction. One example of a triply modified oligonucleotide is also presented, but this requires a more complicated procedure with several different building blocks, i.e., two differently protected-alkyne moieties along with the unprotected alkyne that are then deprotected stepwise. Meyer, et. al., reported the application of TMTA (the thiol Michael-type additions) for monoconjugation of a 5’-thiol oligonucleotide and different acrylamide derivatives (i.e., phenyl, mannose, ferrocene, dansyl, biotin or deoxycholic acid) and also for multiple (up to 2 structurally/functionally different entities per oligonucleotide) conjugation of a tetrathiolbased oligonucleotide and an acrylamide mannose derivative to afford a glycocluster14. The orthogonality of TMTA with copper-catalyzed azide–alkyne cycloaddition (CuAAC) in application to oligonucleotides was also studied. It was demonstrated that the two reactions


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could not be performed together, due to interaction between thiols and copper, but could be performed sequentially to allow the preparation of bis-conjugated oligonucleotides (mannosegalactose, biotin-galactose, biotin-mannose or dansyl-mannose). More recently Pradere and Hall reported a synthesis strategy for the site-specific hetero bislabeling of long RNAs15. This method was employed for the preparation of several Cy3/Cy5 bis-labeled pre-miRNAs in step-wise manner. The first label was introduced by a click reaction (propargyl modified RNA) on solid support at the position closest to the 5’-end (i.e., furthest from the 3’-end) to minimize any influence of the CPG on the conjugation efficiency, and the second label was introduced by click reaction in solution with reverse functionalization (azide modified RNA and alkyne on the label). Methods published to date allow for rather straightforward addition of up to two different entities to the oligonucleotides. Addition of a third conjugate, however, seems to be rather complicated and methods do not allow for even further addition of entities. In contrast, the methodology presented in this paper takes advantage of commercial oligonucleotides that are conjugated in a step-wise manner with three structurally/functionally different entities and also allowing for further conjugations.

Results and Discussion Multiple labeling of oligonucleotides using “click cycles”, where most conjugations can be performed with the oligonucleotide still attached to the solid support, is readily achieved by means of the LMF H-phosphonate (Linker for Multiple Functionalization H-phosphonate 5, Scheme 1). This reagent is synthesized from FmocSerine methyl ester which was first protected with a 4-methoxytrityl group to give 1. Deprotection of the Fmoc to 2 and subsequent aminolysis with 2-(2-aminoethoxy)ethanol gave 3. Further coupling with the preactivated triple bond donor PAMBA (6) gave 4, which upon phosphonylation with diphenyl H-phosphonate gave 5 (Scheme 1). The LMF moiety is part of an independent molecule, that is readily coupled to the 5’terminus of an ON using H-phosphonate coupling. The LMF carries a sufficiently active triple bond for effective Cu(I) catalyzed click reaction and can be attached multiple times to the same ON (due to the MMTr protected primary hydroxyl). Attachment of the LMF is followed by click reaction in a procedure that is essentially cyclic (Scheme 2). This allows for



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preparation of oligonucleotide 5’-conjugates containing at least three different moieties, where each can carry important supplementary functionality. The number of click cycles is potentially unlimited providing the added entities do not interfere with the attachment of subsequent LMFs. MeO HO

MeO MMTrO

O

i

NH

HO O O

MeO MMTrO

ii

NH

O

iii

O

O O

HN NH2

O

MMTrO

O NH2

2

1

HO

3

O OO P H O

HN O

O O

O

O N H

OH

MMTrO

MMTrO

HN

H N

v

6

HN

iv

O

H N

O

O

HN O

4

O

O

O

5

Scheme 1: Synthesis of the Linker for Multiple Functionalization H-phosphonate (LMF Hphosphonate, 5) using PAMBA (6). Reactions conditions: i: 4-methoxytrityl chloride, Py, overnight, r.t.; ii: 30% Et3N in MeCN, overnight, r.t.; iii: 2-(2-aminoethoxy)ethanol, overnight, 45 oC; iv: preactivation of PAMBA with HBTU, NMM in DMF, 0.5 h, r.t. then addition to 3, 2 h, r.t.; v: diphenyl H-phosphonate, Py, 1 h, r.t.

Schematic representation of the “click cycle” is illustrated in Scheme 2, A: The solid supported 5’-hydroxyl oligonucleotide is connected to the LMF using H-phosphonate chemistry, B: deprotection of the primary hydroxyl group allowing for coupling of another LMF moiety (after completion of the click reaction), C: copper catalyzed [3+2] cycloaddition on solid support, D: Start of a new cycle at A or deprotection and cleavage from support. The “click cycle” – (A, B, C and D steps) - can be performed multiple times resulting in an, in principle, unlimited variety of oligonucleotide conjugates both regarding the number of units and their nature. After each completed “click cycle”, depending on the bioconjugate design, the final product can either be cleaved from the support with methanolic ammonia or extended with the next linker unit.


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As a first model ON-constructs utilizing this methodology conjugates were prepared with a short non-charged peptide in N-acetylated form, AcMIF (the tri-amino acid peptide MIF with sequence PLG is known to have blood-brain barrier penetrating properties16). The clickable form (AcMIF-N3, 7, Fig 1) is prepared in just two steps17. Four different syntheses were done, one where only the LMF was attached, one where one cycle (LMF coupling and click attachment of AcMIF) was performed, one where two cycles were performed and one where three cycles were performed. LMF units were attached one by one and each conjugation step was followed by “click” reaction with the AcMIF-N3 (7) peptide. Figure 1A shows the structure of the final compound after three cycles and deprotection, ON construct 8 that carries three AcMIF peptides. The HPLC profile (Figure 1B) illustrates the chromatograms of crude products after: 1) ON1 with only one LMF, 2) ON1-LMF(AcMIF) conjugate after one complete cycle 3) ON1-LMF(AcMIF)-LMF(AcMIF) conjugate after two complete cycles and finally

4)

ON1-LMF(AcMIF)-LMF(AcMIF)-LMF(AcMIF)

(ON-Construct

8),

after

completing three cycles. The identity of the constructs was confirmed by mass spectrometry (ESI-TOF). Thus, the overall procedure with three click cycles resulted in a high conversion to the triply conjugated ON.

Scheme 2: Schematic representation of the “click cycle”. Reactions conditions: i: LMF, PvCl, MeCN/Py (3:1), 5 min, r.t.; ii: I2/Py/H2O, 15 min, r.t.; iii: 3.5% DCA in DCM, 2 min, r.t.; iv: CuI, DIPEA, tBuOH/H2O (1:1), 24 h; v: MeOH/NH3, r.t., 24 h.



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Figure 1: A: Structure of product 8 18mer

2'OMe

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equipped with three linkers for multiple

functionalization and three AcMIF peptides; B: HPLC chromatograms of consecutive steps towards preparation of product 8 (crude products).

The successful synthesis of this model construct encouraged us to prepare oligonucleotide bioconjugates

with

three

different

entities

(Figure

2,

Table

1).

2’-O-

Methyloligoribonucleotides were chosen as these are commonly used model ONs for optimization of conjugation conditions17, 18. These syntheses also demonstrated the versatility of the concept, where several different classes of compounds were incorporated into the triple-entity oligonucleotide conjugates (compounds 9-11, Fig 2). Coupling-click cycles were in general performed on solid supported 18-mer oligonucleotides (ON1 and ON2, see experimental methods for sequences), and in one case on solid support but with the last click reaction performed in solution.


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Table 1: Modified oligonucleotide conjugates 8 - 11. Prod. #

Product name

calc (M)a

found Yieldb Yieldc (M)a (%) (%)

Stepwise Yield (%)d (# conj.)

Stepwise Yield (%)e (# steps)

ON1

ON1-OH

5793

5796

82

-

-

-

8a

ON1-LMF(AcMIF)

6704

6700

71

87

87 (1)

97(4)

8b

ON1-LMF(AcMIF)-

7569

7569

61

75

87 (2)

96 (8)

8433

8434

49

60

84 (3)

96 (12)

LMF(AcMIF) 8

ON1-LMF(AcMIF)LMF(AcMIF)LMF(AcMIF)

ON2

ON2-OH

5826

5825

47d

-

-

-

9

ON2-LMF(AcMIF)-

8424

8424

19

40

73 (3)

93 (12)

8810

8814

12

25

63 (3)

89 (12)

9569

9572

24

50

79 (3)

94 (12)

LMF(sugar

derivative)-

LMF(fluorescent label) 10

ON2-LMF(AcMIF)LMF(sugar

derivative)-

LMF(ASSLNIA peptide) 11

ON2-LMF(AcMIF)LMF(sugar derivative)LMF(m3G 2’OMe AUA-Cap)

a

Recalculated mass of the construct from observed multiply charged ions.

conjugate based on crude chromatogram,

c

b

HPLC yield of final

HPLC yield of all conjugation cycles after neglecting

truncated sequences formed prior to conjugations (due to incomplete coupling during oligonucleotide synthesis). d Estimation of stepwise yields per conjugation steps based on number of conjugations and the overall yield for the conjugations (i.e., from c),

e

Estimation of stepwise yields for all the

conjugation steps based on number of steps and the overall yield for the conjugations (i.e., from c).

Bioconjugate 9 (Figure 2), that contains one peptide, one carbohydrate and one fluorescent moiety, was synthesized using three click cycles on solid supported ON2 first with AcMIF-N3



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(7), then with an azido-equipped mannopyranoside (12) and in the final cycle a fluorescein azide (13) was attached (Figure 2).

Figure 2: Oligonucleotide bioconjugates with three structurally and functionally different entities synthesized using the LMF (HPLC analysis is shown in the supporting information).


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Prepared in a similar manner, construct 10 contains two different peptide entities and one sugar (Figure 2). Besides the AcMIF peptide and a mannose derivative, 10 is also equipped with the muscle-targeting peptide ASSLNIA (14). This peptide has been demonstrated 9- to 20-fold increase in affinity to muscle cells

19, 20

and is therefore a most interesting signal for

ON-based therapeutics focused on diseases of the heart and skeletal muscles, such as muscular dystrophies. The third oligonucleotide bioconjugate, construct 11 (Figure 2), introduces attachment of an additional class of compounds, the nuclear localization signal m3G-Cap (15)12. The first two click cycles were performed as for ON constructs 9 and 10. In this case the m3G-Cap entity was introduced in solution phase after cleaving the ON conjugate from the support subsequent to attachment of the last LMF and before the last click reaction. A clickable m3GCap18, 21, 22 entity was then introduced by performing the click reaction in solution to obtain ON construct 11 (Figure 2).

In conclusion we have developed a linker for multiple functionalization “LMF” and methodology for the synthesis of oligonucleotide bioconjugates carrying several different classes of molecules using “click cycles” on solid support. The methodology allows a high degree of variation of attached entities, which should be most useful for further studies of the combined effects on the biological activity of potential oligonucleotide therapeutics.

Materials and Methods Acetonitrile (MeCN, HPLC grade, VWR), methanol (MeOH, Fisher Scientific), N, N’dimethylformamide (DMF, Merck) were of commercial grade and dried additionally over 3 Å molecular sieves. Dichloromethane (DCM, Fisher Scientific) and pyridine (Py, Merck) and were of commercial grade and dried additionally over 4 Å molecular sieves. Ethyl acetate, hexane, diethyl ether were from Fisher Scientific. Tetrahydrofuran (THF, Fisher Scientific) was distilled from LiAlH4 and used directly after distillation. Silica gel column chromatography was performed on Merck G60, TLC-analysis was carried out on precoated Silica Gel 60 F254 (Merck) with detection by UV light. NMR spectra were recorded on a Bruker AVANCE DRX-400 instrument (400.13 MHz for 1H, 162.00 MHz for MHz for

13

31

P, 100.62

C) and processed with ACD/NMR Processor Academic Edition. The



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oligonucleotide ON1, 2’-OMe-(CCUCUUACCUCAGUUAC)dA [18mer-(2’-OMe-RNAdA)], was assembled in a TWIST™ synthesis column (Glen Research) on an Applied Biosystems 392A DNA/RNA synthesizer using 0.1 M solution of commercial phosphoramidites 2’-OMe-ABz, 2’-O-Me-GiBu, 2’-O-Me-U and 2’-O-Me-CAc in anhydrous acetonitrile and dA-CPG 500 solid support (Glen Research). The oligonucleotide was synthesized using a standard RNA synthesis procedure on a 1.0 µmol scale using 0.3 M 5-benzylthio-1-H-tetrazole (BTT) as activator and 600 s coupling time. The oligonucleotide ON2, 5’-O-DMT-2’-OMeCCUCUUACCUCAGUUACA [18mer-(2’-OMe-RNA)], was purchased from Rasayan Inc. Silica gel column chromatography was performed on Merck G60, TLC-analysis was carried out on precoated Silica Gel 60 F254 (Merck), with detection by UV light. Solid supported peptide ASSLNIA (Rink Amide resin) was obtained from Bachem and functionalized

using

the

Astra

Initiator

Biotage

peptide

Azidoethoxy)ethyl)-2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside

synthesizer. was

(2-(2-

obtained from

MCAT GmbH (Konstanz, Germany) and N-(3-azidopropyl)-3’,6’-dihydroxy-3-oxo-3Hspiro[isobenzofuran-1,9’-xanthene]-6-carboxamide

(6-Fluorescein-Azide)

from

Jena

Bioscience GmbH (Jena, Germany). N-Methyl morpholine (NMM), copper iodide (CuI), trifluoroacetic acid (TFA), triisopropylsilane (TIS) and N,N-diisopropylethylamine (DIPEA) were purchased from Sigma-Aldrich and used without further purification. The following compounds were also obtained commercially and used without further purification: dimethyl sulfoxide (DMSO, Merck), EDTA (Fluka), tert-butanol (tBuOH, Merck), dichloroacetic acid (DCA, Merck), iodine (I2, ACROS), N,N’-diisopropylcarbodiimide (DIC, Iris), ethyl 2cyano-2-(hydroxyimino)acetate (Oxyma, Iris), N-methylpyrrolidone (NMP, Merck Eurolab), N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate

(HBTU)

(Senn Chemicals), sodium hydride (NaH, 60% dispersion in oil, Sigma-Aldrich), bromoacetic acid (Merck Millipore) and propargyl alcochol (Sigma-Aldrich), Pivaloyl chloride (PvCl, Sigma-Aldrich). Methanolic ammonia [MeOH/NH3 (sat)] was prepared by saturating methanol with ammonia gas (AGA gas) at 0 °C and the solution was then stored at -20 °C. 2(2-Azidoethoxy)ethoxyacetic acid, AcMIF-N3 (7) and the azidolinker containing m3G-Cap (15) construct (11) were prepared using known procedures17, 18. Reverse phase HPLC was carried out on a Jasco HPLC system using the Sigma-Aldrich Discovery BIO Wide Pore C18-5, 5µm (250 x 4.6 mm) for oligonucleotide constructs and the Phenomenex Jupiter 4u Proteo 90A (250 x 4.6 mm) for peptide purification, both with 1 mL/min flow rate. The buffers used for reversed phase chromatography were as follows: A:


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50 mM TEAA pH 6.5; B: 50 mM TEAA pH 6.5 in 50% MeCN; C: 0.1% TFA; D: 0.1% TFA in 50% MeCN. Mass spectra (TOF-MS, ES) were obtained using a Micromass LCT electrospray time-of-flight (ES-TOF) instrument.

Methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((4methoxyphenyl)diphenylmethoxy)propanoate (1) Fmoc-Ser-OMe (1 eq., 2.98 mmol, 1.0 g) was coevaporated twice to dryness with dry acetonitrile (MeCN) to remove water. Then, the flask was flushed with N2 and the protected peptide was dissolved in dry pyridine (Py, 29 mL). 4-Methoxytrityl chloride (3 eq., 8.79 mmol, 2.7 g) was added and the reaction was left to stir overnight. The next day, TLC analysis confirmed completion of the reaction. The solution was then concentrated and added toluene (2 x 150 mL) and DCM (2 x 150 mL) was evaporated to remove remaining Py. Next, the crude product was dissolved in DCM (200 mL) and washed with NaHCO3 (aq, 200 mL) 3 times. The organic layer was collected, dried over MgSO4, concentrated under reduced pressure and purified by column chromatography (a linear gradient of ethyl acetate in hexane from 0% to 20%). Fractions containing product were pooled and concentrated giving compound 1 as a colorless oil (1.6 g, 90%). 1H NMR (400 MHz, CD3OD): δ 7.78 –7.76 (2H, m), 7.63 – 7.59 (2H, m), 7.42 – 7.20 (16 ArH, m), 6.83 – 6.81 (2H, m), 5.70 (1H, d, J = 8.6 Hz), 4.51 – 4.49 (1H, m), 3.45 (2H, quintet, J = 10.3, J = 17.8), 4.26 – 4.22 (1H, m), 3.79 – 3.74 (5H, m), 3.61 – 3.58 (1H, m), 3.44 – 3.41 (1H, m). 13C NMR (100 MHz, CDCl3): δ 47.1, 52.5, 54.5, 55.2, 63.7, 67.3, 86.4, 113.2 (2C), 120.0 (2C), 125.1, 125.2, 127.1 (4C), 127.7 (2C), 127.9 (4C), 128.2 (4C), 130.3 (2C), 134.9, 141.3 (2C), 143.81, 143.86, 143.93, 143.97, 155.8, 158.7, 171.1. ES-MS, calc m/z (M+H)+ 636.2362, found 636.2316.

Methyl 2-amino-3-((4-methoxyphenyl)diphenylmethoxy)propanoate (2) Compound 1 (1 eq., 2.12 mmol, 1.3 g) was dissolved in 30 mL of a mixture of 30% triethylamine in acetonitrile and left to stir overnight. The next day, after TLC analysis confirmed completion of the reaction, the acetonitrile was evaporated and the crude product purified by column chromatography (a linear gradient of MeOH in DCM from 0% to 2%). Fractions containing product were pooled and concentrated giving compound 2 as a yellow oil (0.76 g, 92%). 1H NMR (400 MHz, CD3OD): δ 7.41 – 7.39 (4H, m), 7.30 – 7.26 (6H, m), 7.23 – 7.20 (2H, m), 6.84 – 6.82 (2H, m), 3.79 (3H, s), 3.72 (3H, s), 3.58 (1H, t, J = 4.3 Hz), 3.45 – 3.42 (1H, m), 3.37 – 3.34 (1H, m). 13C NMR (100 MHz, CDCl3): δ 52.0, 55.1, 55.2,



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65.5, 86.2, 113.1 (2C), 127.0 (2C), 127.8 (4C), 128.3 (2C), 128.4 (2C), 130.3 (2C), 135.3, 144.16, 144.21, 158.6, 174.7. ES-MS, calc m/z (M+Na)+ 414.1681, found 414.1673

2-Amino-N-(2-(2-hydroxyethoxy)ethyl)-3-((4methoxyphenyl)diphenylmethoxy)propanamide (3) Compound 2 (1 eq., 1.94 mmol, 0.76 g) was dissolved in 1.95 mL of 2-(2aminoethoxy)ethanol and left to stir at 45 oC overnight. The next day, after TLC analysis confirmed completion of the reaction and after the solution attained r.t., the crude product was purified by column chromatography (a linear gradient of MeOH in DCM from 0% to 4%). Fractions containing product were pooled and concentrated giving compound 3 as a colorless oil (0.73 g, 81%). 1H NMR (400 MHz, CD3OD): δ 7.43 – 7.41 (4H, m), 7.31 – 7.20 (8H, m), 6.85 – 6.82 (2H, m), 3.79 (3H, s), 3.70 – 3.67 (2H, m), 3.59 – 3.54 (4H, m), 3.50 3.47 (3H, m), 3.44 – 3.35 (2H, m).

13

C NMR (100 MHz, CDCl3): δ 39.0, 55.2, 55.3, 61.7,

65.7, 70.0, 72.2, 86.6, 113.2 (2C), 127.0 (2C), 127.9 (2C), 128.4 (4C), 130.4 (4C), 135.4, 144.29, 144.32, 158.7, 174.7. ES-MS, calc m/z (M) 463.2233, found 463.2286.

4-((2-(Prop-2-yn-1-yloxy)acetamido)methyl) benzoic acid (PAMBA, 6) A round bottomed flask was charged with DCC (1 eq., 5.3 mmol, 0.6 g), dissolved in 37 mL acetonitrile and chilled in an ice bath. 2-(Prop-2-yn-1-yloxy)acetic acid (16 in ESI, 1 eq., 5.3 mmol, 1.1 g), dissolved in 2 mL of acetonitrile, was added to the reaction mixture and 20 min later, it was followed by 4-(aminomethyl)benzoic acid (1.6 eq., 8.62 mmol, 1.3 g) suspended in 9 mL of acetonitrile. The reaction was left to stir at 0 oC for 1 h, then allowed to attain r.t. and stirred overnight. After TLC analysis confirmed completion of the reaction, the acetonitrile was evaporated and the crude product purified by column chromatography (a linear gradient of MeOH in DCM from 0% to 2%). Fractions containing product were pooled and concentrated giving compound 6 as colorless solid (0.65 g, 50%). 1H NMR (400 MHz, CD3OD): δ 7.99 – 7.97 (2H, m), 7.41 – 7.39 (2H, m), 4.49 (2H, s), 4.29 (2H, d, J=2.01 Hz), 4.11 (2H, s), 2.96 – 2.94 (1H, m). 13C NMR (100 MHz, CDCl3): δ 43.3, 59.5, 69.6, 77.2, 79.8, 128.5 (2C), 131.0, 131.1 (2C), 145.4, 169.8, 172.4. ES-MS, calc m/z (M-H)- 246.0767, found 246.0797.

N-(1-((2-(2-Hydroxyethoxy)ethyl)amino)-3-((4-methoxyphenyl)diphenylmethoxy)-1oxopropan-2-yl)-4-((2-(prop-2-yn-1-yloxy)acetamido)methyl)benzamide (4)


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The reaction flask was charged with PAMBA (6, 1.13 eq., 1.78 mmol, 0.44 g), HBTU (1.1 eq, 1.65 mmol, 0.63 g) and 14 mL DMF. Then NMM (10 eq., 15.7 mmol, 1.73 mL) was added and the reaction was pre-activated for 30 min at r.t. Next, 3 (1 eq., 1.57 mmol, 0.73 g) was added and the reaction was left to stir for 2 h. After MS and TLC analysis confirmed completion of the reaction, the DMF was evaporated under reduced pressure and the crude product purified by column chromatography (a linear gradient of MeOH in DCM from 0% to 3%). Fractions containing product were pooled and concentrated giving compound 4 as a colorless oil (0.61 g, 56%). 1H NMR (400 MHz, CD3OD): δ 7.70 – 7.68 (2H, m), 7.47 – 7.41 (4H, m), 7.35 – 7.29 (8H, m), 7.26 – 7.19 (2H, m), 7.09 – 6.98 (2NH, m), 6.92 (NH, t, J= 5.8 Hz), 6.87 – 6.81 (2H, m), 4.70 (1H, q, J= 4.9 Hz), 4.53 (2H, d, J= 6.1 Hz), 4.23 – 4.23 (2H, m), 4.13 (2H, s), 3.82 – 3.74 (4H, m), 3.65 – 3.60 (2H, m), 3.58 – 3.56 (2H,m), 3.55 – 3.48 (4H, m), 3.32 (1H, dd, J=9.06, 6.04 Hz), 2.50 (1H, t, J= 2.27 Hz).

13

C NMR (100 MHz,

CDCl3): δ 39.5, 42.4, 53.5, 55.2, 58.7, 61.6, 62.9, 64.4, 68.9, 69.4, 72.3, 87.0, 113.3 (2C), 127.1 (2C), 127.5 (2C), 127.8 (2C), 128.0 (2C), 128.2 (4C), 130.2 (2C), 130.2 (2C), 132.6, 134.9, 142.0, 143.8, 144.0, 158.7, 167.0, 169.1, 170.0. ES-MS, calc m/z (M-H)- 692.2972, found 692.2432.

2-(2-(3-((4-methoxyphenyl)diphenylmethoxy)-2-(4-((2-(prop-2-yn-1yloxy)acetamido)methyl)benzamido)propanamido)ethoxy)ethyl phosphonate (LMF Hphosphonate, 5) Compound 4 (1 eq., 0.7 mmol, 0.49 g) was twice dried by evaporation of added dry acetonitrile. Then, the flask was flushed with N2 (gas) and the substrate was dissolved in dry Py (6.5 mL). Diphenyl H-phosphonate (2 eq., 1.4 mmol, 0.27 mL) was added and the reaction was left to stir for 1 h. After TLC analysis confirmed completion of the reaction, 2 mL of water and 0.95 mL of triethylamine was added and the reaction was left to stir for another 1 h. Next, another TLC test was made and after confirming completion of hydrolysis the Py was evaporated. The crude product was dissolved in DCM and washed with NaHCO3 (aq) two times. The organic layer was collected, dried over MgSO4, concentrated and purified by column chromatography (a linear gradient of MeOH in DCM from 0% to 5%). Fractions containing product were pooled and concentrated giving compound 5 as a colorless oil (0.396 g, 74%). 1H NMR (400 MHz, CD3OD): δ 8.50 (1H, m), 7.83 – 7.78 (2H, m), 7.65 – 7.63 (1H, m), 7.46 – 7.41 (4H, m), 7.37 – 7.29 (4H, m), 7.29 - 7. (7H, m), 6.92 – 6.87 (1H, m), 6.82 – 6.77 (2H, m), 6.81 (1H, d, PH, J= 621 Hz), 5.00 – 4.91 (1H, m), 4.54 (2H, d, J= 6.04 Hz), 4.23 (2H, d, J= 2.6 Hz), 4.13 (2H, s), 3.99 – 3.90 (1H, m), 3.77 (3H, s), 3.66 – 3.36 (10H, m),



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2.50 (1H, t, J= 2.5 Hz).

13

C NMR (100 MHz, CDCl3): δ 39.8, 42.4, 45.6, 53.5, 55.2, 58.7,

62.8, 64.3, 69.0, 69.3, 70.9, 86.3, 113.1 (2C), 126.9 (2C), 127.6 (2C), 127.7 (4C), 127.8 (4C), 128.4 (2C), 130.4 (2C), 133.7, 135.4, 141.4, 144.2, 144.3, 158.5, 166.4, 169.0, 170.1. 31

PNMR (161.9 MHz, CDCl3): δ 3.17, 7.01 (1P, dt, J= 621 Hz, J= 11.1 Hz). ES-MS, calc

m/z (M-H)- 756.2686, found 756.2636.

Synthesis of azido functionalized peptide ASSLNIA (N3-ASSLNIA, 14) Solid supported ASSLNIA peptide (1 eq., 90 mg, 0.546 mmol) was placed in a reaction vessel on the peptide synthesizer and the resin was swollen with NMP for 15 min at 70 oC. Next, the Fmoc protecting group was removed at r.t. by addition of 20% piperidine in NMP first for 3 min and then with a second treatment for 10 min. In a separate vial, 2-(2azidoethoxy)ethoxyacetic acid (7.5 eq, 0.369 mmol, 70 mg) was preactivated for 30 min using a 0.2 M solution of Oxyma in NMP (5 eq., 0.245 mmol, 11.7 mg) and a 0.2 M solution of DIC in NMP (5 eq., 0.245 mmol, 12.7 µL). The preactivated solution of azido acid was then added to the resin and the reaction was mixed on the peptide synthesizer for 8 min at 65 o

C (using microwave). After the support was extensively washed the crude product was

cleaved off using a 5 mL TFA/TIS/water (95:2.5:2.5) cocktail (3 h). The TFA was then evaporated and the crude product was then dissolved in water and extracted three times with diethyl ether. The sample was concentrated and purification was done by RP-HPLC using a linear gradient of buffer D in C from 20% to 100% in 37 min, detection at 220 nm and temperature 60 oC. tR = 21.6. ES-MS, calc m/z (M+H)+ 844.4403, found 844.966.

Synthesis of oligonucleotide bioconjugates prepared with linker for multiple functionalization (8-11) All reactions with solid supported oligonucleotides (ONs) were carried out in 2 mL Eppendorf tubes with a screw cap. Each reagent, unless stated differently, was added to the tube with a syringe or pipette, vortexed, centrifuged and the solution above the support was carefully removed using a syringe with a needle. The washing steps following each reaction were carried out in a similar manner. Each ON was extensively washed with MeOH (3 × 0.5 mL), MeCN (2 × 0.5 mL) and DCM (2 × 0.5 mL) before synthesis. Since the procedure for addition of a linker unit and the subsequent copper catalyzed conjugation is the same each time, we decided to describe it as “Cycle”. With this “Cycle 1” mean the addition of the first linker and the “click” attachment of the first entity, “Cycle 2” –


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of the second linker and click conjugation and “Cycle 3” of the third linker and click conjugation.

Synthesis of ON construct 8 – ON1- LMF(AcMIF)-LMF(AcMIF)-LMF(AcMIF) Cycle 1 Solid supported ON1 (2.5 mg CPG supported, 0.1 µmol) was placed in an Eppendorf tube. The support was then washed respectively with DCM (2 × 0.5 mL) and MeCN/Py (3 : 1, 2 × 0.5 mL) to prepare it for introduction of the multiple functionalization linker (LMF). Next, the LMF H-phosphonate 5 was dissolved in anhydrous pyridine (125 µL) and added to support followed by 1.5 eq pivaloyl chloride (PvCl, 2.7 µL) dissolved in anhydrous MeCN (375 µL, 30 mM solution) and the reaction mixture was stirred on a vortex for 5 min. After this time, the coupling solution was removed and the support was washed with MeCN/Py (3 : 1, 3 × 0.5 mL) to ensure removal of remaining reagents. In the following step the solid supported crude product was oxidized with 0.5 mL of a solution of iodine in pyridine/water (1 mL stock solution: 20 mg I2, 980 µL Py, 20 µL H2O) while stirring on a vortex for 15 min. The support was then washed extensively with pyridine/water (9 : 1, 3 × 0.5 mL), MeCN (2 × 0.5 mL) and DCM (2 × 0.5 mL). The MMTr group, protecting the LMF, was then removed with a 3.5 % DCA solution in DCM (2 × 0.5 mL, change of color was observed). The support was subsequently washed with DCM (2 × 0.5 mL) and MeCN (2 × 0.5 mL), to prepare it for the copper catalyzed cycloaddition. The azido-modified MIF peptide (AcMIF-N3 (7),4 eq., 0.158 mg) was dissolved in 50 µL tBuOH/H2O (1 : 1) and added to the support followed by 2 eq. of N,N-diisopropylethylamine (DIPEA) in 25 µL tBuOH/H2O (1 : 1) (from a stock solution of 2 µL/mL; 2eq., 0,0351 µL) and 1 eq. of copper(I) iodide in 25 µL DMSO (from a stock solution of 1.52 mg CuI in 1 mL DMSO) and the reaction was left to stir on a vortex for 24 h. Next day, the conjugation solution was removed and the support washed with tBuOH/H2O (1 : 1, 3 x 0.5 mL), EDTA solution (0.05 mM tBuOH/H2O(7:3), 2 × 0.5 mL), tBuOH/H2O (1 : 1, 2 × 0.5 mL), MeCN (2 × 0.5 mL) and DCM (2 × 0.5 mL). Cycle 2 and 3 The support was washed with MeCN/Py (3 : 1, 2 × 0.5 mL) and the subsequent LMF attachment and click reaction with AcMIF-N3 (7) was performed as in cycle 1 above. To deprotect the final product from solid support, 1 mL MeOH/NH3 (sat) was added and the reaction vial was left on a vortex for 24 h at r.t. After this time, the solution was taken up



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with a syringe and concentrated under reduced pressure. The purification was done by RPHPLC using linear gradient of buffer B in buffer A from 0% to 50% in 50 min, detection at 260 nm and oven temperature 50 oC. tR = 46.16. ES-MS, calc m/z [M]-5 1685.5, found 1685.9. Synthesis of ON construct 9 – ON2-LMF(AcMIF)-LMF(sugar derivative)-LMF(fluorescent label)

Cycle 1 Purchased solid supported ON2 (3 mg CPG supported, 0.1 µmol) was placed in an Eppendorf tube. The subsequent deprotection, washing, LMF attachment and click reaction with AcMIF-N3 (7) was performed as in cycle 1 for ON construct 8. Cycle 2 The support was washed with MeCN/Py (3 : 1, 2 × 0.5 mL) and the subsequent LMF attachment was done as in cycle 1 of construct 8. For the subsequent ”click” reaction (2-(2-azidoethoxy)ethyl)-2,3,4,6-tetra-O-acetyl-alpha-Dmannopyranoside (12, 4 eq., 0.185 mg) was dissolved in 50 µL tBuOH/H2O (1 : 1) and added to the support followed by 2 eq. of DIPEA in 25 µL tBuOH/H2O (1 : 1) (from a stock solution of 2 µL/mL; 2eq., 0,0351 µL) and 1 eq. of copper(I) iodide in 25 µL mixture of DMSO (from a stock solution of 1.52 mg CuI in 1 mL DMSO) and the reaction was left to stir on a vortex for 24 h. Cycle 3 The support was washed with MeCN/Py (3 : 1, 2 × 0.5 mL) and the subsequent LMF attachment was done as in cycle 1 of construct 8. For the subsequent ”click” reaction 6fluorescein-azide (13, 4 eq., 0.184 mg) was dissolved in 50 µL tBuOH/H2O (1 : 1) and added to the support followed by 2 eq. of DIPEA in 25 µL tBuOH/H2O (1 : 1) (from a stock solution of 2 µL/mL; 2eq., 0,0351 µL) and 1 eq. of copper(I) iodide in 25 µL DMSO (from a stock solution of 1.52 mg CuI in 1 mL DMSO) and the reaction was left to stir on a vortex for 24 h.

Deprotection and cleavage from support was performed as for ON construct 8. Purification of 9 was done by RP-HPLC using linear gradient of buffer B in buffer A from 0% to 40% in 40 min, detection at 260 nm and oven temperature 50 oC. tR = 38.3. ES-MS, calc m/z [M]-5 1683.7, found 1683.8.


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Synthesis of construct 10 – ON2-LMF(AcMIF)-LMF(sugar derivative)-LMF(ASSLNIA peptide)

Cycle 1 and 2 Attachment of the first two LMF linked entities was performed as for ON construct 9. Cycle 3 The support was washed with MeCN/Py (3 : 1, 2 × 0.5 mL) and the subsequent LMF attachment was done as in cycle 1 of construct 7. For the subsequent ”click” reaction the azido-modified ASSLNIA-peptide (N3-ASSLNIA (14), 4 eq., 0.338 mg) was dissolved in 50 µL tBuOH/H2O (1 : 1) and added to the support followed by 2 eq. of DIPEA in 25 µL tBuOH/H2O (1 : 1) (from a stock solution of 2 µL/mL; 2eq., 0,0351 µL) and 1 eq. of copper(I) iodide in 25 µL DMSO (from a stock solution of 1.52 mg CuI in 1 mL DMSO) and the reaction was left to stir on a vortex for 24 h.

Deprotection and cleavage from support was performed as for ON construct 8. Purification was performed as for ON construct 9. HPLC tR = 43.4. ES-MS, calc m/z [M]-5 1761.0, found 1761.9.

Synthesis of construct 11 – ON2-LMF(AcMIF)-LMF(sugar derivative)-LMF(m3G 2’OMe AUACap)

Cycle 1 and 2 Attachment of the first two LMF linked entities was performed as for ON construct 8. “Cycle 3” The support was washed with MeCN/Py (3 : 1, 2 × 0.5 mL) and the subsequent LMF attachment was done as in cycle 1 of construct 8. Deprotection and cleavage from support was then performed, as for ON construct 8, prior to the last click reaction. The “click” reaction with the m3G-Cap derivative (15) was then carried out in solution as follows: The azido-modified m3G

2’OMe AUA-Cap

(15, 4 eq., 0.6 mg) was dissolved in 50 µL

tBuOH/H2O (1 : 1) and added to the lyophilized deprotected doubly conjugated ON followed by 2 eq. of DIPEA in 25 µL tBuOH/H2O (1 : 1) (from a stock solution of 2 µL/mL; 2 eq., 0,0351 µL) and 1 eq. of copper iodide in 25 µL DMSO (from a stock solution of 1.52 mg CuI in 1 mL DMSO) and the reaction was left to stir on a vortex for 24 h.



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Purification was performed as for ON construct 9. HPLC tR = 33.1. ES-MS, calc m/z [M]-5 1912.8, found 1913.5

Acknowledgments We gratefully acknowledge funding from Karolinska Institutet and the Swedish Research Council.

Supporting Information NMR and MS spectra for compounds 4 and 5. HPLC chromatograms and MS spectra of all oligonucleotide conjugates

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