Thiol-Cyanobenzothiazole Ligation for the Efficient Preparation of

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Thiol-Cyanobenzothiazole Ligation for the Efficient Preparation of Peptide-PNA Conjugates Nitin Patil, John Karas, Bradley J. Turner, and Fazel Shabanpoor Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00908 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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Bioconjugate Chemistry

Title: Thiol-Cyanobenzothiazole Ligation for the Efficient Preparation of Peptide-PNA Conjugates Nitin A. Patil1, 2, John A Karas3, Bradley J. Turner4, Fazel Shabanpoor4* 1Department

of Microbiology; 2Biomedicine Discovery Institute, Monash University, Clayton 3800, VIC, Australia; 3Department of Pharmacology and Therapeutics, The University of Melbourne, Victoria 3010, Australia; 4The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville 3010, VIC, Australia *Address correspondence to: [email protected]

Abstract Antisense oligonucleotides (ASO)-based drugs are emerging with great potential as therapeutic compounds for diseases with unmet medical needs. However, for ASOs to be effective as clinical entities, they should reach their intracellular RNA and DNA targets at pharmacologically relevant concentrations. Over the past decades various covalently attached delivery vehicles have been utilised for intracellular delivery of ASOs. One such approach is the use of biocompatible cell-penetrating peptides (CPPs) covalently conjugated to ASOs. The stability of the linkage is of paramount importance for maximal intracellular delivery to achieve the desired therapeutic effect. In this study, we have investigated the efficiency and stability of four different bioorthogonal and non-reductive linkages including triazole, thioether, thiosuccinimide thioether and thiazole moieties. Here we have shown that thiazole and thiosuccinimide are the two most efficient and facile approaches for the preparation of peptide-ASO conjugates. The thiazole linkage had a higher stability compared to the thiosuccinimide thioether at physiological conditions (pH 7.4, 37 ºC) in the presence of a biologically relevant concentration of glutathione. We have also showed that the peptide-ASO conjugate with a thiosuccinimide linkage has a significantly lower antisense activity compared to the peptide-ASO with the thiazole linkage, which maintains its antisense activity after 24 hrs of exposure to glutathione. In summary, we have demonstrated that the bioorthagonal thiazole linkage offers the benefits of mild reaction conditions, fast reaction kinetics, absence of any by products and higher stability compared to other conjugation approaches. This facile ligation can be used for the synthesis of a variety of bioconjugates where a stable linkage is required.

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Graphical Abstract

Keywords: conjugation, maleimide, cyanobenzothiazole, click chemistry, peptides, oligonucleotides Introduction Antisense oligonucleotides (ASOs) are typically short oligomers (12-24 monomers) of natural or chemically modified nucleotide analogues that bind to and modulate the function of their cognate mRNA/DNA sequence to elicit a desired pharmacological effect.1 Recently, several new classes of synthetic oligonucleotides with improved physico-chemical and pharmacological properties have been developed. These include

N-(2-aminoethyl)glycyl

peptide

nucleic

acids

phosphorodiamidate morpholino oligonucleotides (PMO),4,

5

(aegPNA),2,

3

2’-O-(methoxyethyl)

oligonucleotides (2’-O-MOE),6 locked nucleic acids (LNA)7 and pyrrolidinyl PNA (acpcPNA).8 This has led to the emergence of new oligonucleotide-based research tools, diagnostics and therapeutics.9 Currently, there are over 80 drug candidates and diagnostics in clinical trials to treat a range of medical conditions, including inflammatory, viral, cardiovascular, ophthalmic and neuromuscular disorders.9,

10

Recently, antisense oligonucleotides such as Eteplirsen© and Nusinersen© (Spinraza)


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have been approved by the FDA for the treatment of Duchenne Muscular Dystrophy (DMD)4 and Spinal Muscular Atrophy (SMA) respectively.6 Despite the enormous therapeutic potential of ASOs, their effective intracellular delivery remains the most significant barrier to clinical translation. This is mainly due to their inability to efficiently penetrate the cellular membrane and reach their intracellular targets. This barrier can be successfully overcome by conjugating ASOs with suitable delivery systems, such as those based on peptides, lipids and carbohydrates.11 Most delivery vectors require covalent attachment to either the 3’- or 5’-end of the ASO.11 Cell-penetrating peptides (CPPs) have shown much promise as delivery vehicles due to their specificity, efficient cell membrane permeability, safety and tolerability. CPP-ASO conjugates have significantly enhanced the intracellular delivery of ASOs, particularly for uncharged morpholino or PNA oligomers.12, 13 They have significantly enhanced the cellular uptake15 and improved in vivo efficacy of ASOs.14, 15 For the synthesis of CPP-ASO conjugates, several methods have been developed.16 Typically, both CPP and ASO precursors are prepared separately via solid-phase synthesis with appropriate functional groups at their termini for the subsequent bio-orthogonal ligation step to form the conjugate. One chemical linkage that has been utilised is the disulfide bond,17, 18 but due to its high sensitivity to the reductive cellular environment, alternatives such as amides,19,

20

thioethers15 and triazoles21,

22

have

often been preferred. Despite their improved stability, the conjugation reactions required to form these chemical moieties can be complicated, inefficient and hence not amenable to scale-up. For example, it is challenging to form an amide bond sitespecifically with peptides bearing lysinyl (Lys), aspartyl (Asp) and glutamyl (Glu) residues without appropriate side-chain protection. This necessitates an extra deprotection step post-ligation and increases the hydrophobicity of the peptide precursor, which makes purification more difficult.23 The copper(I)-catalysed alkyneazide cycloaddition (CuAAC) has also been utilised to form CPP-ASO conjugates due to its chemo-selectivity but it has been shown to have low efficiency with poor conjugation yields.21,

24, 25

In some cases, an organic co-solvent and elevated

temperature have been used to achieve better conjugation efficiency.26, 27



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The thiol-maleimide ligation has found widespread use for the assembly of CPP-ASO conjugates28,

29

due to the ease of incorporation of the reactive functional groups

during solid-phase synthesis of the precursors. The reaction can also be performed under mild physiological conditions and is highly efficient.30,

31

Despite these

advantages, the resultant thioether moiety is still susceptible to reduction in vivo,31, 32 thus leading to premature release of the drug from the delivery vector. Therefore, in this study, we will evaluate three non-reversible, chemo-selective conjugation chemistries including CuAAC25, thiol/halide substitution reaction (thioether)33, 34 and thiazole conjugation35 as alternatives to the thiol-maleimide reaction for the preparation of CPP-ASO conjugates. Results and Discussion Antisense technology is a versatile approach for the development of gene-specific therapies for diseases with unmet medical needs. The recent FDA approval of two antisense oligonucleotides (ASO), Exondys51© and Spinraza© for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy respectively, highlights the great therapeutic potential of this technology. But despite their promise, ASOs suffer from poor pharmacokinetics and inefficient cell membrane permeability.11, 36 The efficient intracellular delivery of peptide-ASO conjugates requires stable and irreversible linkage between the peptide and the ASO. To date, most ligation approaches that been used in the synthesis of peptide-ASO conjugates suffer from inefficiency and lack of stability. Therefore, in the current study, we have explored the 2-cyanothiazole (CBT) conjugation strategy in comparison with thiol-maliemide,15 thiol-chloroacetyl,33,

34, 37

and alkyne-azide click21,

24, 25

as an stable linkage for

preparation of ASO conjugates. In order to prepare the ASO conjugates, we synthesised a 20-mer PNA sequence that targets survival motor neuron-2 (SMN2) pre-mRNA to induce exon-7 inclusion toward producing a full-length SMN2 mRNA and protein.23, 38 The 5’end of the PNA was functionalised with maleimidopropionic acid (PNA-1), 2chloroacetic acid (PNA-2), cysteine (PNA-3) and hexynoic acid (PNA-4) (Figure 1). A miniPEG (polyethylene glycol) spacer was incorporated between the 5’-end of the PNA and the functional moieties to reduce any potential steric hindrance during the 4 ACS Paragon Plus Environment

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conjugation step. All PNAs with appropriate functional groups were cleaved and purified using RP-HPLC to give purities of >95% and yields of between 25-60% (Table 1, Figure S1).

Figure 1: Solid phase synthesis of PNAs: ia) amaleimide propionic acid (2.5 eq.), HCTU (2.2 eq), DIEA (3 eq.), DMF; ib) b2-chloroacetic acid (2.5 eq.), HCTU (2.2 eq), DIEA (3 eq.), DMF; ic) cFmoc-Cys(Trt)-OH (4 eq.), HCTU (3.8 eq), DIEA (4 eq.), DMF; id) d5-hexynoic acid (2.5 eq.), HCTU (2.2 eq), DIEA (3 eq.), DMF; ii) 20% piperidine/DMF; iii) TFA: TIPS: water (95: 2.5: 2.5.

We have chosen a well-known cell-penetrating peptide, ApoE (140-150)23, 39 for the synthesis of Peptide-PNA conjugates. To enable subsequent conjugation with the respective PNA, a cysteine (Peptide-1) or an orthogonally protected lysine (Figure 2B) residue was incorporated at the C-terminus during solid-phase peptide synthesis. Analogues with Lys(ivDde) were further modified with azidoacetyl (Peptide-2) and 2-cyanobenzothiazole (CBT) group (Peptide-3) (Figure 2A) to enable the cycloaddition and thiazole conjugation, respectively. All three analogues were cleaved from solid support and purified by RP-HPLC to greater than 95% purity with a yield of between 40-70% (Table 1, Figure S2). We then investigated the efficiency of each conjugation. Initial attempts to form the triazole conjugate in 50% DMSO at pH 5-6 and ambient temperature for 12 hrs was unsuccessful. Subsequently, the conjugation conditions were optimised by increasing the pH from 5-6 to pH 8 and the temperature to 60°C. These changes allowed the reaction to proceed to completion in 30 mins as confirmed by the disappearance of the HPLC peak corresponding to alkyne-PNA (PNA4) (Figure 3A) with a yield of 56% (C1, Table 1, Figure S3). Although the CuAAC has been utilised widely for preparing 5 ACS Paragon Plus Environment


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bioconjugates,40 we found that high temperature and pH is often necessary to form peptide-PNA conjugates as we have shown previously

41.

These reaction conditions

are not ideal as they can cause degradation of the ASOs. A NC

B S

N

Ac-

ApoE

Trt Ahx Cys

iii

Ac-

SH Ahx Cys

ApoE

Peptide-1 NH2

N3

i

O

iia, iii

Ac-

ApoE

NC N

NH2

Ahx Lys NH2

Peptide-2

S Ac-

ApoE

ivDde Ahx Lys

N

NC

NH O

S

iib, iii O OH

Ac-

O N H

ApoE

O Ahx Lys NH2

Peptide-3

Figure 2: A) Solution phase synthesis of Succinoyl-2-cyanobenzothiazole (CBT). i. succinic anhydride (4 eq.), 4-methylmorpholine (5 eq.), THF. B) Solid phase synthesis of peptides. iia) a3-azidopropionic acid (2.5 eq.), HCTU (2.2 eq), DIEA (3 eq.), DMF; iib) bSuccinoylamido-2cynothiazole (2.5 eq.), HCTU (2.2 eq), DIEA (3 eq.), DMF; iii) TFA: TIPS: water (95: 2.5: 2.5).

For the conjugations involving cysteine, all reactions were carried-out in PBS (pH 7.4). The cysteine-peptide (Peptide-1) was coupled to chloroacetyl-PNA (PNA-2) via a thiolhalide SN2 reaction mechanism to form a thioether moiety (C2, Table 1). The rate of formation of the conjugate was monitored by mass spectrometry. The reaction proceeded slowly but to completion after 12 hrs with a yield of 60% (Figure 3, Table 1, Figure S3). The long incubation time resulted in the formation of peptide dimer and hydrolysis of the chloroacetyl group (Figure 3B). The reaction rate can be accelerated at higher pH but will lead to an increase in the formation of side products which limits the efficiency of this reaction. Therefore, despite the chemo-selectivity of both the CuAAC and thiol-halide reactions, they are not particularly efficient in terms of both reaction time and yield. The thiol-maleimide click reaction has been the most widely used conjugation strategy for the preparation of various bioconjugates. Therefore, we investigated its effectiveness in the preparation of peptide-ASO conjugates. The formation of thiosuccinimide linkages between cysteine in Peptide-1 and maleimide-PNA (PNA-1) was carried out in PBS at pH 7.4 (C3, Table 1, Figure S3). The reaction did not 6 ACS Paragon Plus Environment

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progress any further after 30 mins even in the presence of excess peptide (Figure 3C). It was found that the maleimide moiety had undergone ring-opening hydrolysis as determined by mass spectrometry ([M+H+]+: 5974.1.). The conversion of the maleimide to maleamic acid has rendered it non-reactive towards the thiol group. A recent study has also showed that PEG-maleimide is hydrolytically unstable in an aqueous environment.42 Despite this problem, the formation of the thiosuccinimide linkage was still efficient with a 78% yield (C3, Table 1, Figure S3) and had a faster reaction rate compared to the previous reactions. Table 1. Theoretical and experimental [M+H+]+ molecular masses of the peptide precursors, PNA precursors and peptide-PNA conjugates plus the percentage yields obtained.

Molecular weight Theor. Exp. PNA PNA-1 PNA-2 PNA-3 PNA-4 Peptide Peptide-1 Peptide-2 Peptide-3 Peptide-PNA conjugates C1 C2 C3 C4

% Yield

5955.1 5880.3 5904.8 5899.9

5955.9 5881.4 5904.2 5902.5

25 60 48 50

1609.9 1717.9 1891.5

1610.2 1719.1 1891.9

70 55 40

7618.5 7454.8 7565.1 7779.3

7619.4 7454.2 7566.3 7780.1

56 60 78 89

The final ligation reaction to be evaluated was a thiazole linkage between the succinoyl CBT-peptide (peptide-3) and cysteine-PNA (PNA-4). In comparison to previous reactions, thiazole formation progressed at a much faster rate (complete within 10 mins) as determined by mass spectrometry (Figure 3D, Figure S3). The desired conjugate was obtained in 89% yield, which demonstrates the higher efficiency of the thiol-CBT conjugation chemistry.



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Figure 3: Comparison of conjugation reactions via analytical RP-HPLC. A) Alkyne-azide click conjugation; B) Thiol-halide conjugation; C) Thiol-maleimide conjugation; D) Thiol-CBT conjugation. *desired conjugated product, °starting peptide, ▪ starting PNA, ◊hydrolyzed PNA, ∆peptide dimer.

With confirmation that the thiol-maleimide (C3) and thiol-CBT (C4) reactions are the most efficient, we then sought to assess their stability under physiological conditions using 10 mM GSH to mimic the highly reductive intracellular environment. The C3 and C4 conjugates were incubated in 50 mM phosphate buffer, pH 7.4 (containing 10 mM GSH) at 37°C. The percentage of intact peptide-PNA conjugate was determined over 72 hrs via LC-MS analysis. Both conjugates showed similar stability after 1 hr incubation, however from 6-72 hrs, the level of thiol-maleimide significantly decrease compared to thiol-CBT conjugate. Overall, the thiol-CBT conjugate was found to be more stable to exogenous thiols at physiological condition with 60% of intact peptidePNA conjugate remaining after 72 hrs compared to 20% of intact cysteine-maleimide conjugate (Figure 4). This finding is consistent with previous studies that show the instability of thiosuccinimide thioether linkages.31, 43-45


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Figure 4: LC-MS analysis of thiol-maleimide and thiol-CBT conjugates treated with glutathione (GSH). The level of intact peptide-PNA conjugates were normalised to the level of untreated peptide-PNA conjugates. Data are presented as mean ± SEM for each timepoint with p values

Thiol-Cyanobenzothiazole Ligation for the Efficient Preparation of

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