Next Article in Journal
ZnII and CuII-Based Coordination Polymers and Metal Organic Frameworks by the of Use of 2-Pyridyl Oximes and 1,3,5-Benzenetricarboxylic Acid
Next Article in Special Issue
Conditionally Activated (“Caged”) Oligonucleotides
Previous Article in Journal
Chiral Tertiary Amine Catalyzed Asymmetric [4 + 2] Cyclization of 3-Aroylcoumarines with 2,3-Butadienoate
Previous Article in Special Issue
Synthesis of 5′-Thiamine-Capped RNA
 
 
Communication
Peer-Review Record

Expanding the Scope of the Cleavable N-(Methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates

Molecules 2021, 26(2), 490; https://doi.org/10.3390/molecules26020490
by Aapo Aho, Antti Äärelä, Heidi Korhonen and Pasi Virta *
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Molecules 2021, 26(2), 490; https://doi.org/10.3390/molecules26020490
Submission received: 22 December 2020 / Revised: 8 January 2021 / Accepted: 11 January 2021 / Published: 18 January 2021
(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

Round 1

Reviewer 1 Report

This paper describes synthesis of conjugates constructed as a delivery system for antisense oligonucleotides (AOS) having different bio-targets. The conjugates containing AOS fragment and other biomolecule (oligopeptide, PNA or acetylated 2-amino-2-deoxy-D-galactose construct). The N-
(methoxy)oxazolidine-junction is formed on 2’-deoxy-2’-(N-methoxyamino) uridine molecule. To form oxazolidine ring used oligopeptides, PNA and D-galactosamine construct possess a terminal aldehyde group necessary for final condensation reaction. The conjugates should be delivered to indicated cells and released ON under pH-controlled conditions in the chosen cells. The presented research enters the wide research area seeking for new effective delivery systems for drug transportation across biological barriers. Authors present tedious strategy of conjugates synthesis involving automated synthesis of oligonucleotides and peptides, their purification and finally conjugation via 2’-amino-2’-deoxyuridine.
For D-galactosamine construct another multi steps synthesis was applied. The yields of particular conjugates are moderate. The measurements of hydrolysis time suggest that obtained conjugates really can be applied for pH-controlled releasing of model AOS. In general, the presented idea of conjugates synthesis is interesting and be worth of presentation in the front of scientific community.
Before approval several necessary in my opinion changes should be consider.
Introduction: a sentence” poor dianophoric properties” is unintelligible for me in context of ON applications.
Body text
Aldehydes are usually abbreviated as RCHO. The formula depicted in caption of table 1 should be changed. Please decide about form of half-life time transcript: t0.5 or t0.5.
Used ASO are addressed to different cancer types: AON-ISE –prostate cancer, Nusinersen is used in treatment of SMA, IONIS- 105 DGAT2RX is applied in NASH treatment. On the other hand, the used oligopeptide and D-galactosamine derivatives play also different roles in living systems. Some
comments for used conjugates compositions (oligonucleotide – counterpart) are necessary.
Materials and method
Due to small scale of preparations the information about used amounts of reactants, solvents for washing is necessary, because excess of solvents can provide to yields decreasing.
Line 217 concentration of H2SO4 and amount of H2O should be indicated
Line 221 amount of NaHCO3 solution and DCM have to be indicated
Line 222 the same for brine
Line 230 anhydrous DMF was used or not special dehydrated
Line 233 and 234 amounts of solvents and solutions.
Line 243 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-ᴅ-galactopyranoside is commercially available (manufacturer) or home prepared?
Line 260 introduce space between Celsius grade and its value
Supplementary data
On the beginning of this section the commercially available reagents should be listed with the name of manufacturers, otherwise the references to original preparation should be added.

N-Bz-3-amino-1,1-diethoxypropane
Pyridine anhydrous or used without additional dehydration
You really weighted the benzoyl chloride?

N-Bz-β-Ala-H
N-Bz-3-amino-1,1-diethoxypropane (manufacturer)
Amount of MeCN

N-Fmoc-β-Ala-H
What kind of sodium carbonate has been used?
Amount of NaHCO3 solution using for washing

Figures 3s and 5s have not a time scale

Oxazolidine ß-Ala-H solid support for peptide/PNA aldehyde synthesis (3)
Amounts of used solvents

4. Synthesis of β-Ala-H peptide aldehydes P1 and P2
Amounts of used solvents
DIC with oxyma please change to DIC/Oxyma

Synthesis of PNA1
Amount of mixture of anisole and TFA, Et2O.

7. Small molecule model
An aqueous mixture containing N-Bz-β-Ala-H (5 mM) and the buffer3 , what it means?
Amount of pH buffers used in these experiments

9. Determining hydrolysis rates of ASO-UNOMe conjugates C1, C2, C3, and C4

...were incubated in aq. buffered solution at 37 ± 0.1 °C, what amount of buffer was used?

Figure S16. 1H NMR (600 MHz, CDCl3) spectrum of compound 5.
Carboxylic proton is lost

Author Response

Cf. the loaded response letter.

Author Response File: Author Response.pdf

Reviewer 2 Report

It is very nice study, concerning an important area of oligonucleotide synthesis chemistry, especially in relation to possible biological applications/therapeutic oligonucleotides. Biologically cleavable binding of a cytotoxic molecules is essential especially for successful development of aptamer-drug conjugates as cheaper and more stable alternatives for currently therapeutically used antibody-drug conjugates. Even if this study is a follow-up of the previous one of the authors group, it brings additional significant improving in the binding technology.

I have only couple of notes/question:

Line 105: Just a note - nusinersen (Spinraza®) is approved not only by FDA, but since 2017 also by EMA for use in EU.

Table 1, Figure 2, Table 2: To fully assemble the data, it would be useful to include also the values for pH 7.4, if it is possible to obtain them e.g. from HPLC as shown in Figure 3 and eventually compare them with non-treated oligonucleotide conjugates.

Ad SI, S6, 2. Synthesis of UNOMe-oligonucleotides ON1, ON2, ON3, and ON4

I would like to ensure if really 25% ammonium hydroxide (16h/55 °C) was used for cleavage and deprotection of synthesized ODNs? What G and A protection of exocyclic amino groups were used for synthesis of the ODN?

Author Response

Cf. the loaded response letter.

 

Author Response File: Author Response.pdf

Back to TopTop