Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Synthesis of Fmoc-Protected (S,S)-trans-Cyclopentane Diamine Monomers Enables the Preparation and Study of Conformationally Restricted Peptide Nucleic Acids Hongchao Zheng, Mrinmoy Saha,† and Daniel H. Appella* Synthetic Bioactive Molecules Section, Laboratory of Bioorganic Chemistry (LBC), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, 8 Center Drive, Room 404, Bethesda, Maryland 20892, United States Org. Lett. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 11/21/18. For personal use only.
S Supporting Information *
ABSTRACT: An efficient synthesis of Fmoc-protected (S,S)-trans-cyclopentane PNA (tcypPNA) monomers starting from mono-Boc-protected (S,S)-1,2-cyclopentanediamine is reported. A general synthetic strategy was developed so that tcypPNA monomers with each nucleobase can be made in sufficient quantity and purity for use in solid-phase peptide synthesis (SPPS). The newly synthesized monomers were then successfully incorporated into 10-residue PNA oligomers using standard Fmoc chemistry for SPPS. The different tcypPNAs allow different positions in the sequence to be conformationally constrained with (S,S)-trans-cyclopentane to determine the effects on binding to complementary DNA.
P
eptide nucleic acids (PNAs) 1 are synthetic DNA mimics that consist of nucleobases attached to an acyclic and highly flexible pseudopeptide backbone (Figure 1).1 PNAs bind with high affinity and mismatch selectivity to complementary oligonucleotides.2 The uncharged and non-natural peptide backbone in PNA promotes good binding to DNA and RNA sequences and also conveys resistance to degradation by
enzymes. These unique properties make PNA a promising alternative to oligonucleotide-based molecules for a wide variety of antisense, antigene, and diagnostic applications.3,4 Developing backbone-modified PNAs to increase binding affinity to complementary nucleic acids by suitable preorganization can improve the properties of PNAs, which may subsequently improve both fundamental research and biomedical applications.5 We are developing a strategy to predictably increase the stability and specificity of PNA−oligonucleotide duplexes by incorporation of a cyclopentane ring into the PNA backbone 2 (Figure 1).6 In our previous studies, (S,S)-trans-cyclopentanederived PNAs (tcypPNAs) 2 were successfully prepared from Boc-protected PNA monomers 3 through Boc-based solidphase peptide synthesis (SPPS). In this method, trifluoroacetic acid (TFA) is used to remove the N-terminal Boc group after each amide-coupling step. The final PNAs made by this approach require harsh and toxic conditions (such as HF or triflic acid) to cleave the PNA product from the solid support.6 Currently, most laboratories make peptides using the milder conditions of Fmoc-based chemistry where piperidine is used to cleave the N-terminal Fmoc after each coupling step and TFA is used to cleave the final PNA from the solid support. Many commercial peptide synthesizers will no longer support Boc-based peptide synthesis. PNAs can be successfully made using the Fmoc-based SPPS,7 so an effective method for the preparation of Fmoc-protected tcypPNA monomers 4 is highly desirable to continue studying the structural effects and
Figure 1. Chemical Structures of PNA, tcypPNA, and monomers.
Received: October 22, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b03374 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters applications of tcypPNA oligomers. In this manuscript, we describe an efficient synthesis of Fmoc-protected tcypPNA monomers and demonstrate applications to tcypPNA SPPS and binding studies. The starting material for tcypPNA monomers is mono-Bocprotected (S,S)-1,2-cyclopentanediamine (5). We previously developed several synthetic routes to make enantiopure 5,6a,b but the most efficient approach is via an enzymatic resolution which has allowed the commercialization of 5.9 For this work, commercially purchased 5 with >99% ee was exclusively used. To convert 5 to tcypPNA monomers, we envisioned the most efficient and economical route would be monoalkyation of the primary amine in 5 with a haloacetate ester followed by replacement of Boc with Fmoc. Subsequent attachment of the nucleobase followed by ester deprotection would then afford tcypPNA monomers suitable for Fmoc-SPPS. In our initial synthetic route, a benzyl group (Bn) was selected as the ester protecting group, since it can be removed by catalytic hydrogenolysis (Scheme 1).8 The primary amine 5 underwent
the benzyl ester protecting group and the need to use metal catalysts at any step. A methyl ester protecting group was selected with the idea that it could be potentially removed under mildly basic or enzymatic12 conditions with minimal impact to the basesensitive Fmoc or the acid-sensitive Bhoc in the A, C, and G nucleobases. In the optimized synthesis, additional improvements to the original protocol were also developed (Scheme 2). In the first step, the chiral amine 5 reacts with methyl Scheme 2. Revised Synthetic Route
Scheme 1. Initial Synthetic Route
bromoacetate under basic conditions to provide methyl ester 9 exclusively. The unwanted dialkylation byproduct could be completely eliminated by the slow addition of methyl bromoacetate to a solution of 5 at 0 °C. In the following step, we were concerned that triethylamine could slowly deprotect the Fmoc group and decrease the yield of the product (see step 3 from 6 to 7, Scheme 1).13 Replacing triethylamine with basic, biphasic conditions (NaHCO3 in dioxane/water) to attach the Fmoc group afforded 10 with significantly better yield compared to 7 (Scheme 2). Fmoc protection of the secondary amine was not observed. Next, the coupling of nucleobase carboxylic acids to the amine 10 was performed using the same conditions employed in our initial synthetic route to afford the fully protected cyclopentane PNA monomers 11a−d. Unfortunately, enzymes such as alcalase were not able to hydrolyze the methyl ester. Different enzymes14 were screened for this conversion, and they all failed to give the desired PNA monomers 4. We therefore examined several different basic conditions to determine if the methyl ester can be selectively removed in the presence of Fmoc. Using aqueous conditions around pH 12, methyl ester hydrolysis is complete in 30 min but also leads to some Fmoc deprotection. Performing an additional Fmoc reprotection step after hydrolysis of the methyl ester was the most practical way to obtain the final product. When the methyl esters 11 were subjected to a onepot sequential hydrolysis/Fmoc reprotection, they successfully generated the desired tcypPNA monomers 4, which were used directly in the solid-phase synthesis of PNAs. We have used this synthetic route to produce 4a−d in five steps with 50− 76% overall yields. Despite the extra Fmoc reprotection step, the final tcypPNA monomers are easily obtained in multigram quantities and with high purity. To test the compatibility of the newly synthesized PNA monomers with standard Fmoc-SPPS, 4a−d were incorporated into a 10-residue mixed-base PNA sequence that has been extensively studied in the literature (by Nielsen and coworkers, in particular).15 Four different tcypPNAs 2a−d were
a mostly selective monoalkylation with benzyl bromoacetate in the presence of triethylamine at room temperature to afford benzyl ester 6 along with 10% undesired dialkylation product. The steric hindrance of the cyclopentane ring is most likely responsible for this selectivity. Without purification, crude 6 was treated with TFA to effect the Boc deprotection. The TFA salt was then reacted with 9-fluorenylmethyl N-succinimidyl carbonate (Fmoc-OSu) under basic conditions to provide Fmoc-protected cyclopentane diamine 7 in 54% yield, after purification, over three steps. Combination of thymine 1-acetic acid and chiral amine 7 in the presence of N-ethyl-N′-(3(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and hydroxybenzotriazole (HOBt) formed the fully protected cyclopentane PNA monomer 8. Catalytic hydrogenolysis of benzyl ester 8 yielded Fmoc-protected PNA monomer 4a. This synthetic route produced 4a in five steps from compound 5 with a 44% overall yield. The monomer 4a can be used directly in Fmoc-SPPS of PNAs. However, there were a number of shortcomings with this initial approach that became evident upon scale-up and when expanding to use nucleobases other than thymine. First, the catalytic hydrogenolysis of the benzyl ester had to be closely monitored to avoid unwanted Fmoc deprotection under the reaction conditions.10 Also, the final hydrogenolysis step was incompatible for the synthesis of Fmoc-protected PNA monomers containing A, C, or G nucleobases as the Bhoc protecting group on the exocyclic amines was not stable under the reaction conditions of catalytic hydrogenolysis.11 There were additional concerns about residual metal contamination from the catalysts that affected the final purity of the tcypPNA monomer. To address these issues, we developed a revised synthetic route to avoid B
DOI: 10.1021/acs.orglett.8b03374 Org. Lett. XXXX, XXX, XXX−XXX
Organic Letters
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prepared on a 5 μmol scale using Fmoc-SPPS protocols on an Applied Biosystems 433a automated peptide synthesizer with HATU as the amide-forming reagent.16 After the completion of tcypPNA peptide synthesis, the resin was treated with 5% mcresol in TFA for deprotection and cleavage from the resin. In each PNA, a lysine was present as the first residue and the Nterminal was left unprotected in order to promote aqueous solubility and prevent aggregation. The crude PNAs were readily purified by reversed-phase HPLC (see Supporting Information) and characterized by ESI-TOF mass spectrometry (Table 1).
Letter
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03374. Experimental details and copies of spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Table 1. Mass Characterization Data for All PNAs entrya
sequence
calcd
obsdb
1 2 3 4 5
GTAGATCACT-Lys (1a) GT*AGAT*CACT*-Lys (2a) GTA*GA*TCA*CT-Lys (2b) GTAGATC*AC*T-Lys (2c) G*TAG*ATCACT-Lys (2d)
1428.1 1488.1 1488.1 1468.1 1468.1
1428.1 1488.2 1488.2 1468.2 1468.1
Mrinmoy Saha: 0000-0002-2878-7568 Daniel H. Appella: 0000-0001-7195-7764 Present Address †
(M.S.) Abzena, 360 George Patterson Blvd, Suite 102, Bristol, PA 19007. Notes
The authors declare no competing financial interest.
a
Cyclopentane stereochemistry is (S,S); B* = tcyp residue. All data in this table correspond to the doubly charged ion [M + 2H]2+ of PNAs. b PNAs were characterized using a Waters Xevo-G2 XS qTOFTM instrument. All PNA oligomers gave molecular ions consistent with the final product.
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ACKNOWLEDGMENTS This research was supported by the Intramural Research Program of NIDDK, NIH. We thank John Lloyd (NIDDK/ NIH) for performing mass spectrometry and Robert O’Connor (NIDDK/NIH) for assistance with NMR spectrometry.
The effects of cyclopentane substitution in PNAs were determined by examining the melting temperature for each tcypPNA 2a−d and unmodified PNA 1a when bound to complementary DNA. Incorporation of cyclopentanes in the middle of the PNA sequence consistently increases the melting temperature when bound to complementary DNA (2b and 2c, Table 2). In contrast, the same substitution at the ends of the
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(1) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497. (b) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895. (c) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624. (d) Nielsen, P. E. Curr. Opin. Biotechnol. 2001, 12, 16. (e) Nielsen, P. E. Mol. Biotechnol. 2004, 26, 233. (2) (a) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 9677. (b) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365, 566. (c) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Nordén, B. Nature 1994, 368, 561. (d) Weiler, J.; Gausepohl, H.; Hauser, N.; Jensen, O. N.; Hoheisel, J. D. Nucleic Acids Res. 1997, 25, 2792. (e) Ratilainen, T.; Holmén, A.; Tuite, E.; Nielsen, P. E.; Nordén, B. Biochemistry 2000, 39, 7781. (3) (a) Pandey, V. N.; Upadhyay, A.; Chaubey, B. Expert Opin. Biol. Ther. 2009, 9, 975. (b) Ray, A.; Nordén, B. FASEB J. 2000, 14, 1041. (c) Sardone, V.; Zhou, H.; Muntoni, F.; Ferlini, A.; Falzarano, M. S. Molecules 2017, 22, 563. (d) Ghosal, A. J. Infect. Dev. Countries 2017, 11, 212. (4) (a) Dean, D. A. Adv. Drug Delivery Rev. 2000, 44, 81. (b) Stender, H. Expert Rev. Mol. Diagn. 2003, 3, 649. (c) Gambari, R. Curr. Med. Chem. 2004, 11, 1253. (d) Siddiquee, S.; Rovina, K.; Azriah, A. Adv. Technol. Biol. Med. 2015, 3, 131. (e) Wu, J.; Meng, Q.; Ren, H.; Wang, H.; Wu, J.; Wang, Q. Acta Pharmacol. Sin. 2017, 38, 798. (5) (a) Tedeschi, T.; Sforza, S.; Corradini, R.; Marchelli, R. Tetrahedron Lett. 2005, 46, 8395. (b) Corradini, R.; Sforza, S.; Tedeschi, T.; Totsingan, F.; Manicardi, A.; Marchelli, R. Curr. Top. Med. Chem. 2011, 11, 1535. (c) Rozners, E. J. J. Nucleic Acids 2012, 2012, 518162. (d) Sugiyama, T.; Kittaka, A. Molecules 2013, 18, 287. (e) Moccia, M.; Adamo, M. F.; Saviano, M. Artif. DNA PNA XNA 2014, 5, e1107176. (6) (a) Myers, M. C.; Witschi, M. A.; Larionova, N. V.; Franck, J. M.; Haynes, R. D.; Hara, T.; Grajkowski, A.; Appella, D. H. Org. Lett. 2003, 5, 2695. (b) Pokorski, J. K.; Witschi, M. A.; Purnell, B. L.; Appella, D. H. J. Am. Chem. Soc. 2004, 126, 15067. (c) Englund, E. A.; Xu, Q.; Witschi, M. A.; Appella, D. H. J. Am. Chem. Soc. 2006, 128,
Table 2. Melting Temperature Data for PNA−DNA Complexes entrya
sequence
Tmb (°C)
ΔTmc (°C)
1 2 3 4 5
GTAGATCACT-Lys (1a) GT*AGAT*CACT*-Lys (2a) GTA*GA*TCA*CT-Lys (2b) GTAGATC*AC*T-Lys (2c) G*TAG*ATCACT-Lys (2d)
46.8 60.4 65.2 61.4 53.3
13.6 18.4 14.6 6.5
REFERENCES
a
Cyclopentane stereochemistry is (S,S); B* = tcyp residue. bTm is the melting temperature for PNA−DNA complexes. Conditions for Tm measurement: 154 mM NaCl, 1.55 mM KH2PO4, 5.12 mM Na2HPO4, pH 7.2, UV measured at 260 nm from 15 to 95 °C, in 1 °C increments. All values are averages from three experiments. Approximate error for Tm’s is within 0.9 °C. cΔTm is the difference in melting temperature between unmodified PNA (entry 1) and cyclopentane-modified PNA.
PNA sequence has a smaller effect on the melting temperature since a terminal tcypPNA residue might make a smaller contribution to the total binding affinity in a PNA/DNA duplex (2a vs 2b, 2d vs 2c, Table 2). Taken together, these data indicate that each cyclopentane in the nonterminal position of PNA increases the Tm by an average of 6−7 °C.6 In summary, we describe an efficient synthetic route to Fmoc-protected (S,S)-trans-cyclopentane-derived PNA monomers. The newly prepared PNA monomers are fully compatible with Fmoc-based solid-phase synthesis and could become powerful building blocks for the construction of tycpPNAs with biomedical applications. C
DOI: 10.1021/acs.orglett.8b03374 Org. Lett. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.orglett.8b03374 Org. Lett. XXXX, XXX, XXX−XXX
