Synthesis of novel conjugates of a saccharide, amino acids,nucleobase and the evaluation of their cell compatibility

Article abstract of DOI:10.3762/bjoc.10.250

This article reports the synthesis of a novel type of conjugate of three fundamental biological build blocks (i.e., saccharide, amino acids, and nucleobase) and their cell compatibility. The facile synthesis starts with the synthesis of nucleobase and saccharide derivatives, then uses solid-phase peptide synthesis (SPPS) to build the peptide segment (Phe-Arg-Gly-Asp or naphthAla-Phe-Arg- Gly-Asp with fully protected groups), and later, an amidation reaction in liquid phase connects these three parts together. The overall yield of these multiple step synthesis is about 34%. Besides exhibiting excellent solubility, these conjugates of saccharide-amino acids-nucleobase (SAN), like the previously reported conjugates of nucleobase-amino acids-saccharide (NAS) and nucleobase-saccharide-amino acids (NSA), are mammalian cell compatible.

Full text of DOI:10.3762/bjoc.10.250

Synthesis of novel conjugates of a saccharide, amino acids,
nucleobase and the evaluation of their cell compatibility
Dan Yuan1, Xuewen Du1, Junfeng Shi1, Ning Zhou1, Abdulgader Ahmed Baoum2
and Bing Xu*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 2406–2413.
1Department of Chemistry, Brandeis University, 415 South Street,
doi:10.3762/bjoc.10.250
MS015, Waltham, MA 02453, USA and 2Department of Chemistry,
King Abdulaziz University, Jeddah, Saudi Arabia
Received: 12 June 2014
Accepted: 01 October 2014
Published: 16 October 2014
Email:
Bing Xu* - bxu@brandeis.edu
Associate Editor: K. N. Ganesh
*
Corresponding author
©
2014 Yuan et al; licensee Beilstein-Institut.
Keywords:
License and terms: see end of document.
cell compatibility; nucleobase; peptides; saccharide
Abstract
This article reports the synthesis of a novel type of conjugate of three fundamental biological build blocks (i.e., saccharide, amino
acids, and nucleobase) and their cell compatibility. The facile synthesis starts with the synthesis of nucleobase and saccharide
derivatives, then uses solid-phase peptide synthesis (SPPS) to build the peptide segment (Phe-Arg-Gly-Asp or naphthAla-Phe-Arg-
Gly-Asp with fully protected groups), and later, an amidation reaction in liquid phase connects these three parts together. The
overall yield of these multiple step synthesis is about 34%. Besides exhibiting excellent solubility, these conjugates of
saccharide–amino acids–nucleobase (SAN), like the previously reported conjugates of nucleobase–amino acids–saccharide (NAS)
and nucleobase–saccharide–amino acids (NSA), are mammalian cell compatible.
Introduction
As a result of evolution, nature selects saccharides, peptides, nucleobase–saccharide–amino acids (NSA) [19] are able to self-
and nucleobases as the fundamental building blocks for the assemble to form supramolecular hydrogels and exhibit
creation of biomacromolecules, which lay the molecular foun- promising biological functions, such as promoting the develop-
dations of life. This simple fact and the self-assembly of small ment of zygotes [20]. Moreover, both NAS- and NSA-type
molecules in water have inspired us to explore the conjugates of conjugates exhibit excellent cell compatibility. Except for a few
those three types of building blocks for generating supra- known motifs (naphthalene [21,22], Fmoc [23,24], lipid [25]), it
molecular nanofibers in water [1-15]. Recently, we have is still challenging to judge which molecules could self-
demonstrated that not only the conjugates of nucleobase–amino assemble to form ordered nanostructures. This challenge
acids–saccharide (NAS) [16-18] but also the conjugates of requires the molecular engineering of the conjugates and intense
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study of their properties to provide a molecular basis for the ligand–receptor interactions. The conjugates 5–8, without a
understanding of the fundamental correlation between molec- saccharide segment, have good solubility at acidic conditions,
ular structure and self-assembly.
also present great cell compatibility and dissolve well in water.
These results imply that glucuronic acid is unlikely to be the
Here we investigate new molecular conjugates made of basic key factor for cell compatibility of SAN-type conjugates.
building blocks by connecting a saccharide segment to amino
Results and Discussion
acids, and nucleobase (i.e., saccharide–amino acids–nucleobase,
we term it SAN-type). As shown in Figure 1, in the conjugates Molecular design
1
–4, the saccharide is glucuronic acid, the amino acid segment Figure 1 shows the structures of the designed molecules based
consists of Phe-Arg-Gly-Asp or naphthAla (3-(2-naphthyl)- on the mutation of the sequence of the connection of nucleo-
alanine)-Phe-Arg-Gly-Asp, and the nucleobase is thymine or base, amino acids, and saccharide. To explore the properties of
adenine. Our results show that the conjugates synthesized and the conjugates dependent on the relative positions of the
examined in this work exhibit excellent cell compatibility but building blocks we synthesized conjugates having the following
are unable to self-assemble in water to form hydrogels. Their order: saccharide–amino acids–nucleobase (i.e., SAN-type,
excellent solubility in water implies that these molecules may 1–4). Although oligosaccharide moieties serve as constituents
find applications for breaking up molecular aggregates in water, of glycoproteins in a broad range of cell–cell and cell–matrix
a much-needed property for warranting genuine monomeric recognition events, the introduction of glucuronic acid into
Figure 1: Chemical structures of the saccharide–amino acids–nucleobase conjugates (SAN, 1–4) and amino acids–nucleobase (AN, 5–8).
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peptides at the N-terminus (Figure 1) is rare and worth the O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
exploration [26]. As an amino acid, Phe or naphthAla increases (HBTU)/DIEA to connect the following N-Fmoc amino acid
molecular aromatic–aromatic interactions [19,21,27]. Arg-Gly- (i.e., Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Phe-OH,
Asp, which is a well-established tripeptidic epitope, that modu- Fmoc-3-(2-naphthyl)alanine) by repeating the deprotection and
lates mammalian cell adhesion through binding with integrins coupling steps. At last, we cleaved the pentapeptide 20 with all
on the cell membrane [28,29]. Thymine or adenine, as a unique protected groups from the resin by using trifluoroethanol (TFE)/
types of heteroatom aromatics, not only promote self-assembly DCM (2/8, 2 × 1 hour). The reaction of 20 and NHS/DIC acti-
[
30], but also have the capability for DNA delivery [16]. To vates the carboxyl group on 20 to couple with 12 at pH 8.5,
examine the role of the saccharide, we also designed and which results in 21. The subsequent removal of the Fmoc group
synthesized the respective conjugates of amino acids and provides the protected nucleopeptide 22 [30] (Scheme 3). The
nucleobase without the glucuronic moiety (5–8) for comparison. activation of 19 to react with 22 and the subsequent removal of
Fmoc results in 23. Finally, treatment of 23 with 95% trifluoro-
Synthesis
acetic acid (TFA) for 1 hour and triethylamine (Et3N)/MeOH/
Schemes 1–4 show the syntheses of the SAN conjugates formed H2O 1:4:5) for 2 hours affords conjugate 3 (Scheme 4). By
by the reaction of the amino acid segment with the nucleobase changing the nucleobase and using the similar reaction pro-
and the saccharide derivative. The key steps include N-alkyl- cedures, we obtained the rest of the conjugates shown in
ation, acetylation, solid-phase peptide synthesis (SPPS) and Figure 1.
N-hydroxysuccinimide (NHS)/N,N-diisopropylcarbodiimide
(
DIC)-catalyzed amidation reaction. As demonstrated by the Inability of hydrogelation
example of the synthesis of 3, the use of reported methods [31- Unlike the NAS- and NSA-type conjugates, none of the SAN-
5] (Scheme 1) affords the nucleobase and saccharide deriva- type conjugates form hydrogels at the conditions tested. As
3
tives 12, 16, and 19. We used SPPS [36] (Scheme 2) to synthe- shown in Supporting Information File 1, Figure S1A, B, 1 or 2
size the fully protected naphthAla-Phe-Arg-Gly-Asp (20). After form a viscous aqueous solution at a concentration of 3 wt %
loading the first amino acid (Fmoc-Asp(Ot-Bu)-OH) on the (pH 4.0) after one week aging. Although our previous study
2
-chlorotrityl chloride resin, we blocked the resin by shows that the incorporation of naphthAla, a naphthalene-
dichloromethane (DCM)/methanol (MeOH)/N,N-diisopropyl- containing unnatural amino acid, into NSA is able to induce
ethylamine (DIEA) (8:1.5:0.5), next removed the Fmoc hydrogelation [19], the introduction of naphthAla to 1 or 2,
protecting group by 20% piperidine in N,N-dimethylformamide which makes conjugate 3 or 4, however, is unable to result in
(
DMF), then used the coupling reagent N,N,N’,N’-tetramethyl- hydrogelation (Supporting Information File 1, Figure S2A and
Scheme 1: Synthesis of nucleobase (thymine and adenine) and saccharide (glucuronic acid) derivatives 12, 16, and 19.
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Scheme 2: Solid-phase peptide synthesis of peptide segment Fmoc-naphthAla-Phe-Arg(pbf)-Gly-Asn(Ot-Bu)-OH (20).
Scheme 3: Synthesis of H-naphthAla-Phe-Arg(pbf)-Gly-Asn(Ot-Bu)-adenine (22).
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Scheme 4: Synthesis of saccharide–amino acids–nucleobase conjugate 3.
S2B). The corresponding TEM images showed that conjugates conjugates are cell compatible, indicating that the sequences of
–4 all form aggregates (upon drying) without ordered struc- the three types of building blocks have slight influences to
1
tures. Meanwhile, the removal of the saccharide from the SAN- their cell responses. Based on these results, we speculate
type conjugates, which forms compound 5–8, results in no that the cell compatibility of this type of conjugate is
hydrogel (Supporting Information File 1, Figure S1C, D and not only due to the natural building blocks, but also may origi-
Figure S2C, D). These results suggest that the sequences of the nate from their inability to form aggregates at low concentra-
three types of fundamental biological building blocks in the tions.
conjugates are critical for their self-assembly.
Conclusion
In summary, this article reports the synthesis of novel conju-
Cell compatibility
To investigate the cell compatibility of the conjugates, we incu- gates containing three fundamental biological build blocks
bated compounds 1–8 with HeLa cells [37] for 3 days at a (saccharide, amino acids, and nucleobase) and their cell
concentration range from 20 μM to 500 μM. We compared the compatibility. The attachment of the saccharide to the
cytotoxicity of conjugates 1, 2, 5, and 6 in Figure 2. Com- N-terminal of the peptide or the nucleobase to the C-terminal of
pounds 5 and 6 serve as the control for compounds 1 and 2 the peptide apparently reduces the self-assembly ability of these
since they do not have a saccharide part. The viability of HeLa conjugates in water. Although the atomistic details remain to be
cells incubated with 1, 2, 5, and 6 is around 100% at the study elucidated, the observation itself is intriguing. These results
concentrations for 3 days. Also conjugates 3, 4, 7, and 8 exhibit provide a new understanding of the self-assembly of the conju-
little toxicity to HeLa cells (Figure 3). Particularly, the cytotoxi- gates consisting of fundamental building blocks of biomacro-
cities of conjugates 1, 3–5, and 8 are slightly higher than 100% molecules. One potential application of these SAN-type conju-
at 72 h, it is likely originate from the metabolic activity increase gates is to minimize unwanted aggregates of drug candidates for
in the HeLa cells treated by 1, 3–5, and 8 [38]. These results are increasing the efficacy of drugs, which is a direction worth
consistent with our previous reports that NAS- and NSA-type exploring.
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Figure 2: Cell viability of HeLa cells incubated with (A) 1, (B) 2, (c) 5, (D) 6 at different concentrations for 3 days.
Figure 3: Cell viability of HeLa cells incubated with (A) 3, (B) 4, (C) 7, (D) 8 at different concentrations for 3 days.
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18.Li, X.; Kuang, Y.; Xu, B. Soft Matter 2012, 8, 2801–2806.
Supporting Information
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Supporting Information File 1
General section, experimental section, TEM images of
solutions of 1–8.
1
992–1994. doi:10.1039/c3cc48946a
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[
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Acknowledgments
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This work was partially supported by grant from NIH
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R01CA142746) and a grant from KAU (44-130-35-HiCi). We
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