Conjugation of Oligonucleotides to Peptide Aldehydes via a pH-Responsive N-Methoxyoxazolidine Linker

Article abstract of DOI:10.1021/acs.orglett.0c01815

The formation of N-methoxyoxazolidines in the preparation of oligonucleotide-peptide conjugates was evaluated. The reaction occurred between unprotected 2′-N-(methoxy)amino-modified oligonucleotides and peptide aldehydes in reasonable yields when isolated

Full text of DOI:10.1021/acs.orglett.0c01815

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Letter
Conjugation of Oligonucleotides to Peptide Aldehydes via a pH-
Responsive N‑Methoxyoxazolidine Linker
Aapo Aho, Mika Sulkanen, Heidi Korhonen, and Pasi Virta*
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ABSTRACT: The formation of N-methoxyoxazolidines in the preparation of oligonucleotide−peptide conjugates was evaluated.
The reaction occurred between unprotected 2′-N-(methoxy)amino-modified oligonucleotides and peptide aldehydes in reasonable
yields when isolated. The reaction is reversible under slightly acidic conditions, and it is pH-responsive. The rate and the equilibrium
constant may be varied with structurally different aldehydes, allowing an optimization of the ligation and cleavage rate of the
resultant conjugates. Therefore, this concept can be considered a cleavable linker.
nterest in conjugating oligonucleotides (ONs) with
Ipeptides is currently undergoing a renaissance for the
development of new tools for targeted delivery of ON
therapeutics.1 Of particular interest are peptides that in
conjunction with large macromolecular delivery vehicles (e.g.,
nanoparticles, extracellular vesicles,2 and antibodies3) would
recognize appropriate cell surface receptors and/or facilitate
penetration of ONs through cellular barriers,4,5 which, for
example, is needed for endosomal escape.6 As an example,
antibodies are powerful tools for providing tissue-specific
targeting of ONs7−9 but lead to endosomal entrapment of the
ONs. In addition, membrane-penetrating auxiliaries are
needed, in which certain peptides may be utilized.6−11 Similar
needs may be required in nanoparticle-loaded ONs. While
construction of these multifunctional constructs in the
polydisperse form may be straightforward, synthesis of such
uniform and covalently conjugated constructs is complex. In
these scenarios, you would need an efficient ligation to load
ONs into antibodies or nanoparticles and then another
orthogonal ligation, with nearly similar requirements, to
conjugate ONs to peptides. Furthermore, cleavable linkers
may be required to release the active ON cargo from the
delivery vehicles.12 Ideally, the ligation chemistry itself would
be reversible enough in the intracellular compartments but
stable under physiological conditions, therefore eliminating the
need for extra modifications in the structure to provide these
characteristics. Examples of conventional linkages are disul-
fide13 and hydrazone ligation.14,15 The first reaction is
reversible under the reducing conditions found in the
cytoplasm, and the latter under slightly acidic conditions,
such as in endosomes (pH 5.5−6.2) and tumor tissues.12 Both
reactions are practically orthogonal, allowing robust and
efficient conjugation between appropriately functionalized
unprotected ONs and peptides.
Despite the existing technologies, there is an obvious need
for alternative conjugation chemistries that allow straightfor-
ward and orthogonal ligation between unprotected ONs and
peptides. The plausible biodegradability of the linkage would
be an additional benefit. Furthermore, it would be advanta-
geous if the reactive moieties were readily converted to a solid-
supported reagent. This would enable an automated synthesis
of both reactive constituents (i.e., appropriately modified ONs
and peptides). To meet the needs and requirements
mentioned above, the study presented here describes a novel
conjugation strategy, based on a pH-responsive N-methoxyox-
Received: May 29, 2020
https://dx.doi.org/10.1021/acs.orglett.0c01815
© XXXX American Chemical Society
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azolidine formation between readily available 2′-N-(methoxy)-
amino-modified ONs and peptide aldehydes.
product in Table 1 was also isolated and exposed to the
same conditions, and the reverse reaction was studied. The
decay rates with aliphatic aldehydes (acetaldehyde and
butyraldehyde; t0.5 ∼ 3−5 h at 22 °C) were comparable to
those of hydrazones at pH 5.0,14,15 but the decays with
benzyloxypropionaldehyde and N-Bz-glycinaldehyde were
markedly slower. The formation obeyed a mixed reversible
second- and first-order rate law (cf. eq S3). However, variation
of the experimentally determined reaction rates extracted from
the formation and decay was observed. This may be explained
by partially obfuscating side reactions of the aldehydes.30−32
The reaction with N-Bz-glycinaldehyde was studied further
to determine the optimal conditions for conjugating ONs with
peptide aldehydes. Changing the solvent from H2O to DMSO
improved the equilibrium yield, as expected, but the reaction
rate could be accelerated by mineral salts (NaCl, NaI, and
LiCl).33 Under optimized conditions [5.0 mM 1 with 7.5 mM
N-Bz-glycinaldehyde, 1.5 equiv in a 73:24:3 (v/v/v) DMSO/
AcOH/H2O mixture, 2 M LiCl, 50 °C], the product was
obtained in near quantitative yield (>98%) in 15 min.
The rationale for this design of ligation may be attributed to
a combined chemistry of neoglycosides16 and oxazoli-
dines.17−20 Neoglycosylation is an acid-catalyzed reaction
that occurs between sugar hemiacetals and N-methyl alkoxy-
amines.16 It has been applied to the synthesis of various
carbohydrate conjugates,21−26 due to its orthogonality and
good isosteric similarity to native glycan structures. However,
the equilibrium constant of this reaction is relatively small (in
aqueous solution <100 L mol−1, pH <6).27,28 Upon application
of this same alkoxyamine-promoted acid-catalyzed O,N-
acetalization to form a five-membered oxaozolidine ring,17−20
the equilibrium constant may increase with the same favorable
pH profile of the reaction. To verify this hypothesis, the
reaction with small molecular models was first studied. For this
purpose, 2′-deoxy-2′-(N-methoxyamino)uridine (1) was syn-
thesized from anhydrouridine 3 following the procedure by
Ogawa et al.29 (Scheme 1) and mixed with buffered solutions
Scheme 1. Synthesis of CPG-Immobilized 2′-Deoxy-2′-(N-
methoxyamino)uridine (2) and the 2′-Deoxy-2′-(N-
methoxyamine)-Modified Oligonucleotides
Solid support 2, applicable to an automated synthesis of 2′-
(N-methoxy)amino-modified ONs, was next prepared
(Scheme 1). 5′-O-(4,4′-Dimethoxytrityl)-2′-deoxy-2′-(N-
methoxyamino)uridine (5) was converted to a succinate and
immobilized to a long-chain alkylamino-modified controlled
pore glass (LCAA-CPG, loading of 21 μmol g−1) using a
PyBOP-promoted amide coupling. The unreacted amino
groups and the 3′-OH group on the support were capped by
an acetic anhydride treatment. The Weinreb amide of 2 can be
cleaved by concentrated ammonia, which simplified the
support preparation. To verify this prior to ON synthesis, a
small aliquot of 2 was exposed to concentrated ammonia (5 h
at 55 °C), and the quantitative release of 5 was confirmed by
LC-MS analysis. 2 was then used for the automated assembly
of 2′-deoxy-2′-N-(methoxy)amino uridine (UNOMe)-extended
ONs (Scheme 1): T6UNOMe, AON-ISE-UNOMe (with the
phosphorothioate backbone), and its bicyclononyne derivative,
BCN-AON-ISE-UNOMe (with the native phosphodiester back-
bone). While T6 is just a model, AON-ISE prevents expression
of an androgenic receptor variant (AR-V7) as a potential
therapeutic option for the treatment of castration-resistant
prostate cancer.34 The oligonucleotides were synthesized
under standard conditions on a 1.0 μmol scale using
commercially available phosphoramidite building blocks
(Scheme 1). 3-Phenyl-1,2,4-dithiazoline-5-one (POS) was
used as a sulfurization agent for AON-ISE-UNOMe. Cleavage
with concentrated ammonia, followed by RP HPLC
purification (Figure S7), gave the desired 2′-N-methoxyami-
no-modified oligonucleotides in isolated yields of 32%
(T5UNOMe), 26% (AON-ISE-UNOMe), and 17% (BCN-AON-
ISE-UNOMe). No premature reaction with aldehyde impurities
was encountered during the purification (pH >7). This is in
contrast to what is typical for hydrazine-modified35 or
oxyamine-modified36 oligonucleotides.
of different small molecular aldehydes (Table 1). The reaction
rates and equilibrium constants were determined at pH 4, 5,
and 6 using 5 mmol L−1 1 and each aldehyde at 5 mmol L−1
(at 22 °C). As one can see in Table 1, the reaction behaved
like a neoglycosylation: the reaction was accelerated with a
decrease in pH from 6 to 4, but notably there was a relatively
high equilibrium yield (K = 0.4−9.0 × 103 L mol−1). In each
case, a dynamic equilibrium of R and S stereoisomers of the
oxazolidines was obtained (Figure 1). The reaction rate and
equilibrium constant could be tuned by using different
aldehydes. Relatively fast reaction rates were observed with
aliphatic nonhindered aldehydes (acetaldehyde and butyralde-
hyde; t0.5 ∼ 10 min at pH 4.0). The highest equilibrium
constant was observed with N-Bz-glycinaldehyde (K = 9.0 ×
103 L mol−1, at pH 5.0). In our initial tests (data not shown),
only traces of products were observed with ketones (e.g.,
acetone and levulinic acid) and reaction with conjugated
aldehydes (benzaldehyde, p-methoxybenzaldehyde, pyridine-2-
carboxaldehyde, and cinnamaldehyde) was sluggish. Each
For the synthesis of peptide aldehydes, a published
protocol37,38 was applied to immobilize freshly prepared
Fmoc-Gly-H to L-threonine-modified ChemMatrix resin via
an oxazolidine linkage (Scheme 2). This support and
automatic Fmoc/tBu chemisty were used to assemble two
model peptides: SpyTag-(AEEA)2-Gly-H and THR-Gly-H.
SpyTag peptide, originally discovered by Zakeri et al.,39 binds
irreversibly to its target protein (Spy-Catcher) forming an
isopeptide bond between these peptide−protein components.
B
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Table 1. Formation and Decay of N-Methoxyoxazolidines between 2′-Deoxy-2′-(N-methoxy)amino Uridine and Aldehydes
a
b
c
entry
R
pH
t0.5 formation
t0.5 decay (h)
equilibrium yield (%)
equilibrium constant K (M−1
)
1
2
3
4
5
6
7
8
Me
Pr
4
5
6
4
5
6
4
5
6
4
5
6
7.90 0.27 min
25.8 0.6 min
3.93 0.09 h
11.8 0.6 min
40.1 0.6 min
4.14 0.04 h
4.84 0.02 h
33 1.3 h
12.8 0.6 days
21.6 0.5 min
1.92 0.05 h
25.0 0.7 h
1.54 0.30
3.32 0.40
43.1 2.7
1.50 0.34
5.29 0.58
n/a
16.1 0.7
75.5 16.3
n/a
59
62
67
62
54
49
82
86
74
64
66
693 29
856 27
1211 39
835 49
499
379
7
3
BzNHCH2
4958 50
9038 1265
2224 183
959 28
1144 39
939 34
9
10
11
12
BnOCH2CH2
2.00 0.14
9.98 0.38
88.2 6.8
63
a
b
c
At 22 °C in 50 mM buffer (cf. the Supporting Information). With 5 mM 1 and 5 mM aldehyde. With 5 mM ligation product.
Scheme 3. N-Methoxyoxazolidine Ligation of
a
Oligonucleotides to Peptide Aldehydes
Figure 1. Formation of N-(methoxy)oxazolidine between 1 and N-Bz-
glycinaldehyde. (A) Reaction profile of N-Bz-glycinaldehyde ligation
at pH 4 (Table 1, entry 7). (B) RP HPLC profile of the reaction at 35
h. Compare to the conditions in the Supporting Information.
Scheme 2. Synthesis of Peptide Aldehydes for the
Conjugation of Oligonucleotides
a
(A) An example of an RP HPLC profile of the crude product
mixture (C5). (B) Oligonucleotide release profiles from conjugate C3
at pH 4−7. (C) RP HPLC profiles of the cleavage. ON = AON-ISE-
UNOMe (cf. the conditions in the Supporting Information).
This spontaneous and fast reaction has recently been found to
have many applications in protein engineering. It may be
applied for the development of modern biomaterials, though
immunogenicity issues should be resolved prior to drug
delivery applications.40 THR binds to a transferrin receptor,41
and it has been applied to deliver RNA nanoparticles through
the blood−brain barrier.42,43 The peptides were cleaved from
the support using common TFA/scavenger cocktails (cf. the
Supporting Information), precipitated in Et2O, and purified by
RP HPLC to give the desired peptide aldehydes in 9%
[SpyTag-(AEEA)2-Gly-H] and 11% (THR-Gly-H) isolated
yields.
Three ON−peptide conjugates [C1−C3 (Scheme 3)] were
prepared by mixing the 2′-(N-methoxy)amino-modified ONs
(4 mM T6UNOMe or AON-ISE-UNOMe) with peptide aldehydes
[8 mM, SpyTag-(AEEA)2-Gly-H or THR-Gly-H, 2 equiv] in a
1:3 (v/v) AcOH/DMSO mixture and 2 M LiCl. The mixtures
were incubated at 50 °C for 1 h, purified by RP HPLC (Figure
S12), and lyophilized to dryness to give the desired ON−
petide conjugates as white powders in 41% (C1), 12% (C2),
and 28% (C3) isolated yields. No premature degradation of
the N-methoxyoxazolidine linkage occurred during HPLC
purification or lyophilization. According to MS(ESI), no
depurination or marked S → O conversion of the
phosphorothioates44 was detected under the ligation con-
ditions. One bis-peptide−ON conjugate was also prepared by
exposing BCN-AON-ISE-UNOMe first to strain-promoted
azide−alkyne cycloaddition with an azide-modified endosomal
escaping peptide (N3-AEEA-Phe-Trp-Phe-NH2, to yield
conjugate C4)6 and then to N-methoxyoxazolidine formation
(experimental details in the Supporting Information). A larger
10 equiv excess of SpyTag-(AEEA)2-Gly-H was used to give
the desired bis-peptide−ON conjugate (C5) in 38% isolated
yield (Scheme 3A).
C
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Finally, deconjugation rates were studied by incubating the
conjugate (C3, 10 μM) in a buffered solution at pH 4, 5, 6, and
7 and at 37 °C. HPLC and MS analysis of the released payload
matched with the unconjugated AON-ISE-UNOMe (but with
partial desulfurization). Consistent with the small molecule
model studies (Table 1), C3 was fully degraded in 2 weeks at
pH 5 and 4 weeks at pH 6, whereas only 11% and 25% of the
ON was released in 2 and 4 weeks, respectively, at pH 7
(Scheme 3B,C).
REFERENCES
■
(1) Dowdy, S. F. Nat. Biotechnol. 2017, 35, 222.
(2) Yerneni, S. S.; Lathwal, S.; Shrestha, P.; Shirwan, H.;
Matyjaszewski, K.; Weiss, L.; Yolcu, E. S.; Campbell, P. G.; Das, S.
R. ACS Nano 2019, 13, 10555.
(3) Dovgan, I.; Koniev, O.; Kolodych, S.; Wagner, A. Bioconjugate
Chem. 2019, 30, 2483.
(4) Oller-Salvia, B.; Sanchez-Navarro, M.; Giralt, E.; Teixido, M.
Chem. Soc. Rev. 2016, 45, 4690.
(5) McClorey, G.; Banerjee, S. Biomedicines 2018, 6, 51.
́
́
In conclusion, the reversible N-methoxyoxazolidine linkage
was found to be a viable option for the preparation of ON−
peptide conjugates. This reaction occurred readily under
slightly acidic conditions between 2′-N-(methoxy)amino-
modified ONs and peptide aldehydes. The 2′-N-(methoxy)-
amino-modified ONs needed for the conjugation are readily
available by using an automated synthesis and an appropriate
solid support. Small molecule examinations demonstrated that
the reaction favors aliphatic nonhindered aldehydes. The rate
and the equilibrium constant of the reaction may be varied
with structurally different aldehydes. The reaction is reversible
and dynamic under slightly acidic conditions, whereas the
products showed only marginal decay at pH 7. Therefore, the
reaction showed on/off characteristics. The decay rates proved
to be slow, considering the idea of the cleavable linker, but the
data derived from small molecule examinations suggest that
there are other options for peptide aldehydes, allowing an
optimization of the cleavage rate, and the ligation itself.
̈
(6) Lonn, P.; Kacsinta, A. D.; Cui, X.-S.; Hamil, A. S.; Kaulich, M.;
Gogoi, K.; Dowdy, S. F. Sci. Rep. 2016, 6, 32301.
(7) Shi, S.-J.; Wang, L.-J.; Han, D.-H.; Wu, J.-H.; Jiao, D.; Zhang, K.-
L.; Chen, J.-W.; Li, Y.; Yang, F.; Zhang, J.-L.; Zheng, G.-X.; Yang, A.-
G.; Zhao, A.-Z.; Qin, J.-W.; Wen, W.-H. Theranostics 2019, 9, 1247.
(8) Cuellar, T. L.; Barnes, D.; Nelson, C.; Tanguay, J.; Yu, S.-F.;
Wen, X.; Scales, S. J.; Gesch, J.; Davis, D.; van Brabant Smith, A.;
Leake, D.; Vandlen, R.; Siebel, C. W. Nucleic Acids Res. 2015, 43,
1189.
(9) Arnold, A. A.; MAlek-Adamian, E.; Le, P. U.; Meng, A.;
MArtinez-Montero, S.; Petrecca, K.; Damha, M. J.; Shoichet, M. S.
Mol. Ther.–Nucleic Acids 2018, 11, 518.
(10) Takayama, K.; Hirose, H.; Tanaka, G.; Pujals, S.; Katayama, S.;
Nakase, I.; Futaki, S. Mol. Pharmaceutics 2012, 9, 1222.
(11) Rydberg, H. A.; Matson, M.; Amand, H. L.; Esbjorner, E. K.;
Norden, B. Biochemistry 2012, 51, 5531.
(12) Bargh, J. D.; Isidro-Llobet, A.; Parker, J. S.; Spring, D. R. Chem.
Soc. Rev. 2019, 48, 4361.
(13) Chu, B. C. F.; Orgel, L. Nucleic Acids Res. 1988, 16, 3671.
(14) Ollivier, N.; Olivier, C.; Gouyette, C.; Huynh-Dinh, T.; Gras-
Masse, H.; Melnyk, O. Tetrahedron Lett. 2002, 43, 997.
(15) Zatsepin, T. S.; Stetsenko, D. A.; Arzumanov, A. A.; Romanova,
E. A.; Gait, M. J.; Oretskaya, T. S. Bioconjugate Chem. 2002, 13, 822.
(16) Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269.
(17) Fife, T. H.; Hutchins, J. E.C. J. Org. Chem. 1978, 100, 7620.
(18) Walker, R. B.; Huang, M.-J.; Leszczynski, J. J. Mol. Struct.:
THEOCHEM 2001, 549, 137.
(19) Johansen, M.; Bundgaard, H. J. J. Pharm. Sci. 1983, 72, 1294.
(20) Moloney, G. P.; Iskander, M. N.; Craik, D. J. J. Pharm. Sci.
2010, 99, 3362.
(21) Ishida, J.; Hinou, H.; Naruchi, K.; Nishimura, S.-I. Bioorg. Med.
Chem. Lett. 2014, 24, 1197.
̈
́
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01815.
Experimental details for the synthesis of 2, 2′-N-
methoxy-modified oligonucleotides, peptide aldehydes,
and conjugates C1−C5 and reaction profiles and
characterization of the N-methoxyoxazolidines in Table
1 (PDF)
(22) Langenhan, J. M.; Endo, M. M.; Engle, J. M.; Fukumoto, L. L.;
Rogalsky, D. R.; Slevin, L. K.; Fay, L. R.; Lucker, R. W.; Rohlfing, J.
R.; Smith, K. R.; Tjaden, A. E.; Werner, H. M. Carbohydr. Res. 2011,
346, 2663.
AUTHOR INFORMATION
Corresponding Author
■
Pasi Virta − Department of Chemistry, University of Turku,
20014 Turku, Finland; orcid.org/0000-0002-6218-2212;
Email: pamavi@utu.fi
́
(23) Teze, D.; Dion, M.; Daligault, F.; Tran, V.; Andre-Miral, C.;
Tellier, C. Bioorg. Med. Chem. Lett. 2013, 23, 448.
(24) Bohorov, O.; Andersson-Sand, H.; Hoffmann, J.; Blixt, O.
Glycobiology 2006, 16, 21C.
Authors
(25) Huang, M. L.; Cohen, M.; Fisher, C. J.; Schooley, R. T.;
Gagneux, P.; Godula, K. Chem. Commun. 2015, 51, 5326.
Aapo Aho − Department of Chemistry, University of Turku,
20014 Turku, Finland
Mika Sulkanen − Department of Chemistry, University of
Turku, 20014 Turku, Finland
Heidi Korhonen − Department of Chemistry, University of
Turku, 20014 Turku, Finland
́
(26) Clo, E.; Blixt, O.; Jensen, K. J. Eur. J. Org. Chem. 2010, 2010,
540.
̈
(27) Osterlund, T.; Korhonen, H.; Virta, P. Org. Lett. 2018, 20,
1496.
(28) Baudendistel, O. R.; Wieland, D. E.; Schmidt, M. S.; Wittmann,
V. Chem. - Eur. J. 2016, 22, 17359.
(29) Ogawa, A.; Tanaka, M.; Sasaki, T.; Matsuda, A. J. Med. Chem.
1998, 41, 5094.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c01815
(30) Kalia, J.; Raines, R. T. Angew. Chem., Int. Ed. 2008, 47, 7523.
́
(31) Gambaryan, N. P.; Kaitmazova, G. S.; Kagramanova, E. M.;
Notes
Simonyan, L. A.; Safronova, Z. V. Bull. Acad. Sci. USSR, Div. Chem. Sci.
1984, 33, 1012.
(32) Moon, J. B.; Hanzlik, R. P. Biochim. Biophys. Acta, Protein Struct.
Mol. Enzymol. 1987, 914, 1.
(33) Wang, S.; Nawale, G. N.; Kadekar, S.; Oommen, O. P.; Jena, N.
K.; Chakraborty, S.; Hilborn, J.; Varghese, O. P. Sci. Rep. 2018, 8,
2193.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The financial support from the Academy of Finland (308931)
and Business Finland (448/31/2018) is acknowledged.
D
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Organic Letters
pubs.acs.org/OrgLett
Letter
(34) Luna Velez, M. V.; Verhaegh, G. W.; Smit, F.; Sedelaar, J. P.
M.; Schalken, J. A. Oncogene 2019, 38, 3696.
(35) Zatsepin, T. S.; Gait, M. J.; Oretskaya, T. S.; Stetsenko, D. A.
Tetrahedron Lett. 2006, 47, 5515.
(36) Salo, H.; Virta, P.; Hakala, H.; Prakash, T. P.; Kawasaki, A. M.;
̈
Manoharan, M.; Lonnberg, H. Bioconjugate Chem. 1999, 10, 815.
(37) Ede, N. J.; Bray, A. M. Tetrahedron Lett. 1997, 38, 7119.
(38) Ede, N. J.; Eagle, S. N.; Wickham, G.; Bray, A. M.; Warne, B.;
Shoemaker, K.; Rosenberg, S. J. Pept. Sci. 2000, 6, 11−18.
(39) Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-
Linek, U.; Moy, V. T.; Howarth, M. Proc. Natl. Acad. Sci. U. S. A.
2012, 109, No. E690.
(40) Wang, W.; Liu, Z.; Zhou, X.; Guo, Z.; Zhang, J.; Zhu, P.; Yao,
S.; Zhu, M. Nanomedicine 2019, 16, 69.
(41) Lee, J. H.; Engler, J. A.; Collawn, J. F.; Moore, B. A. Eur. J.
Biochem. 2001, 268, 2004.
(42) Prades, R.; Guerrero, S.; Araya, E.; Molina, C.; Salas, E.; Zurita,
́
́
E.; Selva, J.; Egea, G.; Lopez-Iglesias, C.; Teixido, M.; Kogan, M. J.;
Giralt, E. Biomaterials 2012, 33, 7194.
́
́
(43) Oller-Salvia, B.; Sanchez-Navarro, M.; Giralt, E.; Teixido, M.
Chem. Soc. Rev. 2016, 45, 4690.
̈
(44) Oivanen, M.; Ora, M.; Almer, H.; Stromberg, R.; Lonnberg, H.
J. Org. Chem. 1995, 60, 5620.
E
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