PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Am Chem Soc. Author manuscript; available in PMC 2010 November 18.
Published in final edited form as:
PMCID: PMC2789006
NIHMSID: NIHMS155722

Oligocarbonate Molecular Transporters: Oligomerization Based Syntheses and Cell Penetrating Studies

Abstract

An external file that holds a picture, illustration, etc.
Object name is nihms155722f6.jpg

A new family of guanidinium-rich molecular transporters featuring a novel oligocarbonate backbone with 1,7-sidechain spacing is described. Conjugates can be rapidly assembled irrespective of length in a one step oligomerization strategy that can proceed with concomitant introduction of probes (or by analogy drugs). The new transporters exhibit excellent cellular entry as determined by flow cytometry and fluorescence microscopy, and the functionality of their drug delivery capabilities was confirmed by the delivery of the bioluminescent small molecule probe luciferin and turnover by its intracellular target enzyme.

New strategies, devices and agents that enable or enhance the passage of drugs or probes across biological barriers are required to address a range of major challenges in chemotherapy, imaging, diagnostics, and mechanistic chemical biology.1 In 2000, we reported that the cellular uptake of the Tat49–57 peptide could be mimicked by homooligomers of arginine.2 Uptake was shown to be a function of the number and array of guanidinium groups, observations that led to the design and synthesis of the first guanidinium-rich (GR) peptoids,2 GR-spaced peptides,3 GR-oligocarbamates4 and GR-dendrimeric molecular transporters (MoTrs).5 Noteworthy subsequent studies from several groups showed that a variety of other scaffolds, including beta-peptides, carbohydrates, heterocycles, and peptide nucleic acids, upon perguanidinylation, exhibit cell-penetrating activity.6 GR MoTrs have been shown to carry a variety of cargos into cells, including small molecules, probes, metals, peptides, proteins, siRNA, morpholino-RNAs, and DNA plasmids.7 Activatable MoTrs have been reported for targeted therapy and imaging,8 a releasable octaarginine-drug conjugate has been shown to overcome Pgp-mediated resistance in animal models of cancer,9 and a drug-heptaarginine conjugate has been advanced to phase II human clinical trials.10

While octaarginine MoTrs have been made on scale under GMP conditions and a step-saving segment doubling approach has been introduced,11 the length and associated costs of these syntheses preclude some anticipated applications. A solid phase synthesis of octaarginine requires ≥16 steps, while the segment doubling approach involves 9 steps.11 We report herein a new family of oligocarbonate GR MoTrs that can be flexibly and efficiently assembled in a one-step organocatalytic ring opening oligomerization process that also allows for concomitant probe (or drug) attachment and control over transporter length.

We have previously shown that a metal-free, organocatalytic ring-opening polymerization (ROP)12 of cyclic carbonates13 initiated by a variety of nucleophiles, including alcohols, amines and thiols, provides narrowly dispersed polymers of predictable molecular weights and end-group fidelity.14 We reasoned that if cyclic carbonates incorporating a guanidinium side chain could be used in this process, and if the initiator could be a drug or probe, then one-step assembly of oligocarbonate MoTr-drug or -probe conjugates could be realized. Significantly, unlike solid or solution phase syntheses of oligomeric MoTrs in which step count increases with transporter length, this controlled catalytic oligomerization strategy would provide access to various lengths in one step simply through adjustment of the initiator-monomer ratio.15 Moreover, the metal-free nature of the catalysts and low catalyst loadings (typically 5%) are anticipated to avoid the cytotoxicity associated with catalyst residues.

The new guanidine-protected monomer 3 was prepared by coupling the cyclic carbonate 1 and 1,3-di-Boc-2-(2-hydroxyethyl) guanidine 2. It is noteworthy that alcohol 2 does not initiate oligomerization of monomer 3 in the absence of catalyst. However, when the alcohol-tagged dansyl fluorophore initiator 4a or protected sulfur alcohol 4b (Scheme 1) is mixed with monomer 3 in the presence of the bifunctional thiourea/amine catalyst TU/DBU,16 ring opening oligomerization readily occurs. This catalyst exhibits exquisite selectivity for ring-opening oligomerization; no transesterification is observed. This exquisite control stems from the high selectivity of this catalyst combination towards the strained cyclic carbonate of the monomer relative to the acyclic carbonate and ester moieties of the oligomers.14,16 Moreover, oligomers of various lengths are generated by simply controlling the monomer-to-initiator ratio ([M]o/[I]o). Oligomers 5a–e exhibit well defined molecular weights and narrow polydispersities (Mn = 3,800, 5,200, 10,000, 3,900, 5,100; Mw/Mn = 1.16, 1.11, 1.15, 1.16, 1.16, respectively). With a 5 mol % catalyst loading ([M]0 = 1M), full conversion is reached in 1.25 h at room temperature. The process is highly reproducible over the range of scales studied (50mg to 2.5g). 1H NMR spectroscopy showed that each oligomer was end-labeled with the initiator, and the overlay of the GPC traces from the RI and UV detectors confirms quantitative initiation and predictable molecular weights (see SI). Removal of the Boc groups by simple exposure of 5a–e to TFA gave oligocarbonate MoTr conjugates 6a–e in high yields from 3.

Scheme 1
Oligomerization Strategy

The new MoTr conjugates 6a–e incorporate a backbone scaffold (carbonate) and side chain spacing (1,7) previously unexplored in cell uptake studies.3 A distinguishing feature of these molecular transporters is their stability profile; while they are stable for months as solids at room temperature or in buffer (PBS) at ≤4°C, they degrade under physiologically relevant conditions (Hepes buffered saline, pH 7.4) with a half-life of ~8 h at 37°C. This affords excellent shelf stability, but also the novel ability to degrade after cellular uptake. Additionally, the MoTrs are non-toxic at concentrations required for uptake analysis (5 min incubation, EC50 6a=160µM; 6b=48µM, for additional toxicity and stability information see SI). Like analogous oligoarginines, these transporters are highly water-soluble, but as shown for 6a and 6b, they readily partition into octanol when treated with sodium laurate (1.2 equiv. per charge) (SI).17

The ability of GR oligocarbonate MoTrs 6a–c to enter cells was initially determined by flow cytometry with Jurkat cells that had been incubated for 5 min at 23°C with the dansylated oligomers, washed with PBS to remove the remaining oligomers, and resuspended in PBS for analysis (Figure 1). The uptake of 6a–c was compared to that of a dansylated octaarginine derivative (r8, see SI for synthesis) as a positive control and the dansyl initiator 4a as a negative control using the same 5 min pulse strategy.

Figure 1
Flow cytometry determined cellular uptake of oligocarbonates 6a and 6b, dansylated-r8, and dansyl initiator 4 in either PBS or high [K+] PBS. Jurkat cells were incubated with the various transporters or positive and negative controls for 5 minutes at ...

The dansyl probe initiator 4a alone does not enter Jurkat cells. In striking contrast, dansyl-oligocarbonate conjugates 6a and 6b exhibited rapid and concentration-dependent uptake similar to that of the dansylated r8 positive control. The extended oligomer 6c showed uptake but also cell-cell adhesion behavior and was excluded from further analysis (SI). The significant increase in uptake observed for 6b relative to 6a at higher concentrations is consistent with the increase in uptake observed for MoTrs with increasing guanidinium content (up to n=15)18 and provides further evidence that the GR-oligocarbonates are functionally analogous to oligoarginines.

Not unlike the behavior of other GR MoTrs, the uptake of 6a and 6b was drastically decreased when cells were incubated with modified PBS in which all sodium ions were replaced with potassium ions (Figure 1), a protocol used to decrease the voltage potential across the cell membrane.17 Additionally, incubating cells with NaN3, conditions known to interfere with ATP dependent processes,19 led to a decrease in uptake (SI). Finally, decreased uptake (18–37%) was observed with cells incubated at 4°C, suggesting a mixed mechanistic pathway in which endocytosis could play a role (SI).20

In addition to flow cytometry studies, fluorescence microscopy using a two-photon excitation method established that both 6a and 6b were internalized into Jurkat cells upon incubation for 5 minutes at 23°C (Figure 2).

Figure 2
Fluorescence microscopy images showing internalization of 6b throughout various layers (0.9 µm wide) of a Jurkat cell (5 min incubation, 25 µM at 23°C). Panels A, G, L and O show a series of z-cuts through the cell as illustrated ...

To further probe the ability of the oligocarbonate MoTrs to function as delivery vectors, experiments examining the delivery of the bioluminescent small molecule luciferin were conducted. In this recently introduced assay,21 the ability of a conjugate to enter cells and release its luciferin cargo is measured by the light emitted when luciferin is converted by luciferase to oxyluciferin and a photon of detectable light. Only free luciferin is measured and the analysis is independent of the mechanism(s) of entry, providing a real-time measure of drug/probe availability. A new strategy to access thiol-terminated oligomers 6d and 6e (Scheme 1, SI) enabled the facile synthesis of disulfide-releasable luciferin conjugates 7a and 7b (Figure 3). The ability of 7a and 7b to deliver luciferin into HepG2 cells expressing click beetle luciferase was analyzed with a cooled charge-coupled device camera (photon count). Importantly, alkylated luciferin is not a substrate for the luciferase enzyme,22 and all light observed is therefore derived from the intracellular release and turnover of free luciferin.

Figure 3
Assay for measurement of intracellular luciferin delivery.

Figure 4 shows the uptake and delivery of luciferin for 7a, 7b, an analogous D-cysteine-r8 conjugate,21 and luciferin alone in Ringers (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM D-glucose, 2 mM MgCl2, and 2 mM CaCl2) and high [K+] Ringers (70 mM NaCl, 75 mM KCl, 10 mM HEPES, 10 mM D-glucose, 2 mM MgCl2, and 2 mM CaCl2) solutions, imaging buffers which contain a variety of ions and glucose to maintain healthy cells during longer imaging times. Following a 5 min incubation of the luciferase-expressing cells with transporter, both oligocarbonate MoTr conjugates 7a and 7b continuously release free luciferin over a period of about one hour. This behavior is in contrast to the r8 control, which exhibits much faster release kinetics both in cells (over about 20 minutes, Figure 4), and when treated with DTT in buffer, as observed by analytical HPLC analysis (see SI). The ability of these MoTrs to release cargo over time offers several advantages, including the potential to avoid bolus effects associated with administration of a free drug alone. The oligocarbonate MoTrs are able to deliver free luciferin in a concentration-dependent manner (SI) that is inhibited by high [K+] conditions associated with a diminished membrane potential. Free luciferin alone, while marginally cell permeable, exhibits negligible light output after a 5 min incubation. Taken together, these data demonstrate that the novel oligocarbonate MoTrs are able to not only penetrate the cell membranes of multiple cell types, but also efficiently deliver and release small molecule cargos where they are available for turnover by intracellular targets.

Figure 4
Observed bioluminescence from HepG2 cells expressing click beetle luciferase following a 5 minute incubation with 25µM 7a, 7b, cysr8 luciferin derivative, or luciferin alone in either Ringers or [K+] Ringers solutions.

In conclusion, an expedient one-step, metal-free oligomerization route to a new family of MoTrs is described. This strategy enables the direct conjugation of probes and, by analogy, drug moieties as part of the oligomerization process. The monomers could thus be used as “kit” reagents for transporter-conjugate synthesis. Importantly, these oligocarbonate MoTrs show low cytotoxicity and exhibit uptake comparable to or better than that of the parent oligoarginines as determined by flow cytometry and fluorescence microscopy. In addition, their ability to intracellularly deliver and release the bioluminescent small molecule probe luciferin was demonstrated, confirming the intracellular availability of the free cargo to interact with its target enzyme. The facile cellular uptake exhibited by these new MoTrs, the ease with which short to long oligomers (and presumably mixed oligomers) can be prepared, and their ability to degrade after uptake offer many advantages for drug/probe delivery, particularly for biological and macromolecular cargos.

Supplementary Material

1_si_001

Acknowledgment

This work was supported in part by the National Institutes of Health (CA31841 and CA31845 to P.A.W.), the Center on Polymeric Interfaces and Macromolecular Assemblies (NSF-DMR-0213618), NSF-GOALI Grant (NSFCHE-0645891), and fellowship support from the NSF (B.M.T. and M.K.K.), Stanford University (B.M.T.) and Eli Lilly (C.B.C.). We thank the Chris Contag group at Stanford University for facilities and support for the biological experiments.

Footnotes

Supporting Information Available: Experimental procedures, flow cytometry and concentration dependent uptake data, NMR data and fluorescence microscopy images. This material is available free of charge via the internet at http://pubs.acs.org.

References

1. a) Wender PA, Galliher WC, Goun EA, Jones LR, Pillow TH. Adv. Drug Del. Rev. 2008;60:452–472. [PMC free article] [PubMed] b) Snyder EL, Dowdy SF. Expert Opin. Drug Del. 2005;2:43–51. [PubMed] c) Langel U. Cell-Penetrating Peptides: Processes and Applications. Boca Raton, FL: CRC Press; 2002.
2. Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. Proc. Natl. Acad. Sci. U. S. A. 2000;97:13003–13008. [PubMed]
3. Rothbard JB, Kreider E, VanDeusen CL, Wright L, Wylie BL, Wender PA. J. Med. Chem. 2002;45:3612–3618. [PubMed]
4. Wender PA, Rothbard JB, Jessop TC, Kreider EL, Wylie BL. J. Am. Chem. Soc. 2002;124:13382–13383. [PubMed]
5. Wender PA, Kreider E, Pelkey ET, Rothbard J, VanDeusen CL. Org. Lett. 2005;7:4815–4818. [PubMed]
6. For a review and lead references on GR transporters from the groups of Torchilin, Prochiantz, Langel, Futaki, Vives, Wender, Dowdy, Piwnica-Worms, Lebleu, Seebach, Gellman, Goodman, Tor, Chung, Kiso, Mendoza and others see: Adv. Drug Delivery Rev. 2008;60:452. [PMC free article] [PubMed] For further lead references see a) Hamilton SK, Harth E. ACS Nano. 2009;3:402–410. [PubMed] b) Geisler I, Chmielewski J. J. Chem. Biol. Drug Des. 2009;73:39–45. [PubMed] c) Seow WY, Yang Y-Y. Adv. Mater. 2009;21:86–90. d) Daniels DS, Schepartz A. J. Am. Chem. Soc. 2007;129:14578. [PubMed]
7. For a recent review on arginine-rich peptides and their many cargos see: Tung CH, Weissleder R. Adv. Drug Delivery Rev. 2003;55:281–294. [PubMed]
8. a) Goun EA, Shinde R, Dehnert KW, Adams-Bond A, Wender PA, Contag CH, Franc BL. Bioconjugate Chem. 2005;17:787–796. [PubMed] b) Jiang T, Olson ES, Nguyen QT, Roy M, Jennings PA, Tsien RY. Proc. Natl. Acad. Sci. U.S.A. 2004;101:17867–17872. [PubMed]
9. Dubikovskaya EA, Thorne SH, Pillow TH, Contag CH, Wender PA. Proc. Natl. Acad. Sci. USA. 2008;105:12128. [PubMed]
10. Rothbard J, Garlington S, Lin Q, Kirschberg T, Kreider E, McGrane P, Wender P, Khavari P. Nat. Med. 2000;6:1253. [PubMed]
11. Wender PA, Jessop TC, Pattabiraman K, Pelkey ET, VanDeusen CL. Org. Lett. 2001;3:3229–3232. [PubMed]
12. Kamber NE, Jeong W, Waymouth RM, Pratt RC, Lohmeijer BGG, Hedrick JL. Chem. Rev. 2007;107:5813–5840. [PubMed]
13. Rokicki G. Prog. Polym. Sci. 2000;25:259–342. (b) Al-Azemi TF, Bisht KS. Macromolecules. 1999;32:6536–6540.
14. a) Pratt RC, Nederberg F, Waymouth RM, Hedrick JL. Chem. Comm. 2008:114–116. [PubMed] (b) Nederberg F, Lohmeijer BGG, Leibfarth F, Pratt RC, Choi J, Dove AP, Waymouth RM, Hedrick JL. Biomacromolecules. 2007;8:153–160. [PubMed]
15. Guanidinylated oligomers have been generated by ring-opening metathesis to provide cell or artificial membrane transporters with hydrocarbon backbones; unlike our approach, these methods utilize a post-oligomerization functionalization step to introduce the guanidine functionality or fluorescent tags. See Kolonko EM, Kiessling LL. J. Am. Chem. Soc. 2008;130:5626–5627. [PubMed] Kolonko EM, Pontrello JK, Mangold SH, Kiessling LL. J. Am. Chem. Soc. 2009;131:7327–7333. [PubMed] Gabriel GJ, Madkour AE, Dabkowski JM, Nelson CF, Nusslein K, Tew GN. Biomacromolecules. 2008;9:2980–2983. [PubMed] Hennig A, Gabriel GJ, Tew GN, Matile S. J. Am. Chem. Soc. 2008;130:10338–10344. [PubMed]
16. (a) Pratt RC, Lohmeijer BGG, Long DA, Lundberg PNP, Dove AP, Li H, Wade CG, Waymouth RM, Hedrick JL. Macromolecules. 2006;39:7863–7871. (b) Dove AP, Pratt RC, Lohmeijer BGG, Waymouth RM, Hedrick JL. J. Am. Chem. Soc. 2005;127:13798–13799. [PubMed]
17. Rothbard JB, Jessop TC, Lewis RS, Murray BA, Wender PA. J. Am. Chem. Soc. 2004;126:9506–9507. [PubMed]
18. Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB. J. Peptide Res. 2000;56:318–325. [PubMed]
19. Sandvig K, Olsnes S. J. Biol. Chem. 1982;257:7504–7513. [PubMed]
20. a) Silhol M, Tyagi M, Giacca M, Lebleu B, Vives E. Eur. J. Biochem. 2002;269:494. [PubMed] b) Lee H-L, Dubikovskaya EA, Hwang H, Semyonov AN, Wang H, Jones LR, Twieg RJ, Moerner WE, Wender PA. J. Am. Chem. Soc. 2008;130:9364–9370. [PubMed]
21. Jones LR, Goun EA, Shinde R, Rothbard JB, Contag CH, Wender PA. J. Am. Chem. Soc. 2006;128:6526. [PubMed]
22. Denburg JL, Lee RT, McElroy WD. Arch Biochem Biophys. 1969;134:381. [PubMed]