Synthesis of second-and third-generation asp oligopeptide conjugated dendrimers for bone-targeting drug delivery

Article abstract of DOI:10.1080/00397910902883660

The second-and third-generation multiple dendrimers based on naproxen in core and Asp(4-6) oligopeptides in periphery were synthesized in a convergent approach and well characterized by NMR and mass spectral (MS) techniques. These conjugates showed a high affinity to hydroxyapatite in vitro and provided an effective entry for the synthesis of a peptide dendrimer used for bone targeting.

Full text of DOI:10.1080/00397910902883660

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Synthesis of Second- and Third-
Generation Asp Oligopeptide
Conjugated Dendrimers for
Bone-Targeting Drug Delivery
Liang Ouyang a , Junzhu Pan a , Yu Zhang a & Li Guo a
a Key Laboratory of Drug Targeting and Drug
Delivery System of Education Ministry, Department
of Medicinal Chemistry, West China School of
Pharmacy, Sichuan University, Chengdu, China
Published online: 16 Oct 2009.
To cite this article: Liang Ouyang , Junzhu Pan , Yu Zhang & Li Guo (2009) Synthesis
of Second- and Third-Generation Asp Oligopeptide Conjugated Dendrimers for
Bone-Targeting Drug Delivery, Synthetic Communications: An International Journal
for Rapid Communication of Synthetic Organic Chemistry, 39:22, 4039-4052, DOI:
10.1080/00397910902883660
To link to this article: http://dx.doi.org/10.1080/00397910902883660
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Synthetic Communications1, 39: 4039–4052, 2009
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910902883660
Synthesis of Second- and Third-Generation Asp
Oligopeptide Conjugated Dendrimers
for Bone-Targeting Drug Delivery
Liang Ouyang, Junzhu Pan, Yu Zhang, and Li Guo
Key Laboratory of Drug Targeting and Drug Delivery System of
Education Ministry, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu, China
Abstract: The second- and third-generation multiple dendrimers based on
naproxen in core and Asp(4–6) oligopeptides in periphery were synthesized in a
convergent approach and well characterized by NMR and mass spectral (MS)
techniques. These conjugates showed a high affinity to hydroxyapatite in vitro
and provided an effective entry for the synthesis of a peptide dendrimer used
for bone targeting.
Keywords: Asp oligopeptide, bone targeting, convergent synthesis, hydroxyapa-
tite, peptide dendrimer
INTRODUCTION
Dendrimers are synthetic, highly branched, spherical, monodisperse
macromolecules. One of the important developments in this field of
pharmacy is the synthesis of drug-targeting delivery systems.[1] Constructs
bearing multiple copies of a target moiety often display dramatically
increased affinity and specificity relative to the monomer as a result
of avidity and cooperativity effects. Dendrimers are promising as
drug-delivery vehicles also by virtue of their multivalency, well-defined
Received November 18, 2008.
Address correspondence to Li Guo, Key Laboratory of Drug Targeting and
Drug Delivery System of Education Ministry, Department of Medicinal Chemis-
try, West China School of Pharmacy, Sichuan University, Chengdu 610041,
China. E-mail: rosaguoli2000@yahoo.com.cn
4039

4040
L. Ouyang et al.
and large globular structure, mono- or low polydispersity, and
amenability to postsynthetic manipulation.[2] To date, various types of
dendrimers have been investigated as drug-targeting delivery systems.
Examples include polyamidoamine (PAMAM), poly(propylene imine)
(PPI), and poly(lysine).[3] This strategy has been applied to problems in
cancer imaging, cancer therapy, and gene delivery.[4]
Dendrimers conjugated with biologically active peptides have proved
to have increased activities.[5] Asp oligopeptides are novel bone-targeting
moieties with four to six consecutive negatively charged amino acid resi-
dues that readily adsorb certain calcium-containing surfaces and exhibit a
remarkable affinity to bone-hydroxyapatite. (The fully protected Asp(4–
6) 1a–c is shown in Fig. 1.[6] Unlike the P-C-P bond of bisphosphonates,
the oligopeptides are biologically labile and enzymatically degraded. The
advantageous property can make for more efficient drug release, and
there is no unexpected long-term effects of the bone-targeting moiety.[7]
Figure 1. Structure of the fully protected Asp(4–6) (1a–c) and the dendrimer core
(2,3).

Synthesis of Peptide Dendrimers
4041
We aim to increase the bone-targeting potency of the peptides with a
dendritic approach and prepare sphere-type monodispersed Asp oligo-
peptide conjugated dendrimers with larger surface[8] as drug-carrying
candidates for bone targeting.
In the current article, we report the design and synthesis of the
second- and third-generation dendritic compounds with four or eight
Asp(4–6) fragments. They may provide a potential opportunity to study
the influence of shape and architecture or numbers of Asp oligopeptides
on biodistribution, pharmacokinetics, and bone targeting through
systematically adjusting the generation of the dendrimers. We employ
iminodipropionic acid derivatives as novel scaffolds, and the structure of
these dendrimer cores are shown in Fig. 1. Naproxen was used for a model
drug for two reasons: first, naproxen is a clinically relevant anti-
inflammatory agent helpful for treating osteoarthritis; second, it is amen-
able to synthetic manipulations (through amidation of the b-carboxy
group) that provide a mechanism for conjugation and release. The dendri-
mer molecules linked by labile and hydrolyzable peptide bonds target the
osseous tissue, where the active oligopeptides are easily cleavable, and the
removal of the dendrimer periphery groups, liberation of the terminal
amides, and subsequent unzipping of the dendrimer scaffold lead to release
of the parent drug naproxen.[9] A hydroxyapatite (HAP) binding assay was
established under in vitro conditions, and the affinity of the targeting
conjugates for HAP is reported here. Preliminary study of drug release,
biodistribution, and pharmacokinetic analysis of these conjugates for
delivering naproxen into the bone in vivo is now under way.
RESULTS AND DISCUSSION
Preparation of Protected Bone-Targeting Peptides
The fully protected bone-targeting peptides 1 were synthesized by a
conventional liquid-phase peptide synthetic method from Boc-Asp-Obzl
utilizing IBCF (isobutyl chloroformate) and NMM (N-methyl morpho-
line) (mixed anhydrides method) by a series of segment condensation.
The -COOH part dissolved in anhydrous tetrahydrofuran (THF) was
cooled at ꢀ15ꢁC (using a cryohydrate bath (ice=NaCl ¼ 3:1, w=w), and
1 eq. NMM and IBCF were added and stirred for 5 min. The -NH2 part
in THF was added to the reaction mixture. After the coupling reaction
was completed at room temperature, the mixture was purified by recrys-
tallization or silica-gel column chromatography using CH2Cl2 and
CH3OH (100:1–60:1, v=v) as an eluent and deprotected in TFA
(trifluoroacetic acid) to give the peptides 1a–c.

4042
L. Ouyang et al.
Synthesis of the Dendrimer Core
Bis-addition of benzylamine to ethylacrylate through a Michael addition
reaction, then debenzylation and coupling to Boc-Gly-OH or naproxen
through an amide linkage using IBCF=NMM, came next. At last, the
protecting groups were removed in NaOH=CH3OH to provide the
dendrimer cores 2 and 3.
Synthesis of the Peptide Dendrimers
A series of peptide dendrimers were prepared through a convergent
synthesis. The synthetic route is outlined in Scheme 1. We chose iminodi-
propionic acid derivatives as the central core for the polyamide dendri-
mers.[10] However, the reactivity of the imine group was weak, and
therefore in high steric hindrance, the amido linkage with the carboxylic
group could not be formed with a coupling reagent. For the convenience
of synthesis of the dendrimers, a glycine residue was previously intro-
duced using more active IBCF=NMM (mixed anhydride) as coupling
reagent.
We previously synthesized the first-generation dendrimers using
DCC=Hobt (dicyclohexylcarbodiimide=1-hydroxybenzotriazole).[11] How-
ever, this method has some defects: for example, DCU (dicyclohexylurea)
is difficult to eliminate completely, and the purity of the crude product is
poor. In the present research, a water-soluble carbodiimide (EDCIfꢂ HCl)
was used: the yield raised from 50 to 70%, and the workup procedure was
simple. In a similar convergent strategy, the second-generation dendrimers
bearing four oligopeptides (Ia–c) were synthesized after deprotection of
4a–c with trifluoroacetic acid (TFA). EDCI=Hobt was used as a good
coupling reagent; the yield was up to 60%. In the synthesis of the
third-generation dendrimers bearing eight oligopeptides IIIa–c, using
EDCI ꢂ HCl did not give the product in acceptable yields. Several other
coupling reagents (DCC, IBCF=NMM, CDI, etc.) gave similar poor
results. Only the solid-phase peptide coupling reagent HBTU=DIPEA
(o-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate=N,N-
diisopropylethylamine) afforded the products in good yields. We belive
that the poor yields were due to the large size of the reactant. Once the
first chain was attached to the scaffold, the steric bulk inhibited
attachment of additional chains.[12] Conversion of Ia–c and IIIa–c
into the corresponding Asp oligopeptides (IIa–c and IVa–c) was
accomplished by hydrogenolysis over 10% palladium on charcoal; the
reactions required only filtration and evaporation to provide isolated
products.

Synthesis of Peptide Dendrimers
4043
Scheme 1. Reagents and conditions: i) 1a–c, EDCI ꢂ HCl, Hobt, CH2Cl2; ii) TFA,
CH2Cl2; iii) 2, EDCI ꢂ HCl, Hobt, DMF; iv) 3, EDCI ꢂ HCl, Hobt, CH2Cl2; v) H2,
Pd=C (10%, wt%), CH3OH; vi) 2, HBTU, Hobt, DIPEA, DMF.
The high degree of symmetry in these dendrimers enabled facile
confirmation of both structure and purity by NMR techniques. For
1
example, in the H NMR spectrum of dendron 4a–c, the core protons
observed the resonance signals at 1.44 (s), 2.36 (t), and 3.53 (t) ppm were

4044
L. Ouyang et al.
clearly distinguishable from the resonances arising from the wedges (Asp
oligopeptides) at 2.71 (m) and 4.84 (m) ppm. Integration of the respective
areas of the core protons and b-methine protons of Asp oligopeptides
confirmed the complete coupling of the central core 2 and peptides 1.
For the second-generation and third-generation dendrimers IIa–c and
IVa–c, although some resonance signals were not well resolved, the com-
plete cascade reactions could be verified through comparison of integra-
tion areas of the core protons at 2.57 (brs), 3.41 (brs), and 7.32–7.87 (m)
ppm and and b-methine protons of Asp oligopeptides 2.77 (brs) ppm.
Furthermore, the structures of these dendrimers were further verified
by electrospray ionization–mass spectrometry (ESI-MS) or matrix-
assisted laser desorption ionization time-of-flight mass spectrometer
(MALDI-TOF MS). All of the spectra displayed a very prominent peak
corresponding to the dendrimers complexed with protons or sodium
cation. Moreover, elemental analysis was also in good agreement with
those of the signed structures.
Binding of Conjugates to Hydroxyapatite (HAP)
To demonstrate the direct effect of molecular weight and Asp oligopeptide
content on the binding of the conjugates, the in vitro binding of the conju-
gates on HAP was studied. The results of the in vitro binding of the peptide
dendrimer conjugates to HAP are shown in Table 1. The efficiency of bind-
ing was expressed as the percentage of the original amount of conjugate
bound to HAP in dependence of the weight ratio HAP=conjugate.
The results from these in vitro assay suggest several trends. First, all
conjugates were very effcient at binding HAP, being taken up at>60%
and generally >70% over 2 h, whereas the parent drug naproxen was at
best negligibly bound. Second, as expected, a greater content of Asp
Table 1. UV spectra of the conjugates
Peptide
dendrimer
Before HAP
addition (mg=mL)
After HAP
addition (mg=mL)
Ratio (%) of
binding dendrimer
IIa
IIb
IIc
IVa
IVb
IVc
Naproxen
100
100
100
100
100
100
100
32.6
37.2
25.2
26.4
28.6
30.2
99.3
67.4
62.8
74.8
73.6
71.4
69.8
0.7a
aNaproxen dissolved in anhydrous CH3OH.

Synthesis of Peptide Dendrimers
4045
oligopeptide or longer chain of Asp acid in an oligopeptide resulted in
higher binding than the low-molecular-weight and low-Asp-content
conjugate, but no difference in the binding of high or low Asp
oligopeptide content conjugates was observed. Most probably, the size of
the conjugate prevented efficient access to the surface (pores) of HAP.[13]
In conclusion, we have prepared a range of second and third-
generation monodisperse peptide dendrimer–naproxen conjugates
bearing four or eight Asp oligopeptide sequences as peripheral groups
for testing as novel potential bone-targeting therapeutics. The 1H
NMR and MS results support the formation of the targeted dendrons
and dendrimers. Steric hindrance played an important role, and it was
difficult to obtain higher generation dendrimers in a reasonable yield.
We are continuing to explore higher generations of dendritic molecules
that bearing Asp sequences and develop other routes to increase both
the number of conjugated drug molecules in a controllable method and
applications to other bioactive molecules.
EXPERIMENTAL
General
All reactions requiring anhydrous conditions were performed under an
Ar or N2 atmosphere. Chemicals and solvents were either analytical-
reagent grade or purified by standard techniques. Thin-layer chromatogra-
phy (TLC) used silica-gel plates GF254; compounds were visualized by
irradiation with ultraviolet (UV) light and=or by treatment with a solution
of phosphomolybdic acid (20% wt. in ethanol) followed by heating. Column
chromatography was performed using silica gel with eluent given in parenth-
eses. 1H NMR analysis was performed using CDCl3 or D2O as a solvent at
room temperature. The chemical shifts are expressed in relative to tetra-
methylsilane (TMS, ¼ 0 ppm) and the coupling constants J are given in hertz.
Hydroxyapatite (HAP) was purchased from Shanghai Institute of Biochem-
istry with surface area of 9.12 m2=g and average particle size of 15mm.
General Procedure for the Synthesis of the Dendron 4
The protected oligopeptides NH2-Asp(4–6) 1 (2.0 equiv), compound 3 (1.0
equiv) and 1-hydroxybenz otriazole, Hobt (2.2 equiv) were dissolved in
anhydrous THF. N1-((ethylimino)methylene)-N3, N3-dimethylpropane-
1,3-diamine hydrochloride, and EDCI ꢂ HCl (2.2 equiv) were added into
the solution.The reaction mixture was stirred under N2 at room tempera-
ture for 24 h. The solution was then concentrated under vacuum, and the

4046
L. Ouyang et al.
crude product was purified on a silica-gel chromatography column using
CH2Cl2 and CH3OH (100:1–40:1, v=v) as eluent.
Data
Compound 4a
Nankin wax (0.48 g, 68%) from 3 (0.1 g, 0.31 mmol). 1H NMR (400 MHz,
CDCl3): 1.44 (s, 9 H, Boc-H), 2.42 (t, 4 H, J ¼ 6.0 Hz, CH2CO ꢃ 2),
2.71–3.04 (m, 16 H, Asp-bCH2), 3.52 (t, 4 H, J ¼ 6.0 Hz, N-CH2 ꢃ 2),
3.85 (s, 2 H, Gly-CH2), 4.73–4.84 (m, 8 H, Asp-aCH), 5.1 (brs, 20 H,
ph-CH2), 7.25 (brs, 50 H, ph-H). ESI MS (m=z): calcd. for 2162.8
([MþNa]þ); obsd. 2162.9.
Compound 4b
1
Yellow oil (0.50 g, 62%) from 3 (0.1 g, 0.31 mmol). H NMR (400 MHz,
CDCl3): 1.42 (s, 9 H, Boc-H), 2.52 (t, 4 H, J ¼ 6.4 Hz, CH2CO ꢃ 2),
2.70–2.91 (m, 20 H, Asp-bCH2), 3.54 (t, 4 H, J ¼ 6.4 Hz, N-CH2 ꢃ 2),
3.88 (s, 2 H, Gly-CH2), 4.71–4.92 (m, 10 H, Asp-aCH), 5.14 (brs, 24 H,
ph-CH2), 7.21 (brs, 60 H, ph-H). ESC MS (m=z): calcd. for 2549.9
([MþH]þ), obsd. 2550.3.
Compound 4c
White wax (0.67 g, 73%) from 3 (0.1 g, 0.31 mmol).1H NMR (400 MHz,
CDCl3): 1.44 (s, 9 H, Boc-H), 2.58 (t, 4 H, J ¼ 6.4 Hz, CH2CO ꢃ 2),
2.72–3.01 (m, 24 H, Asp-bCH2), 3.58 (t, 4 H, J ¼ 6.4 Hz, N-CH2 ꢃ 2),
3.92 (s, 2 H, Gly-CH2), 4.69–4.82 (m, 12 H, Asp-aCH), 5.09 (brs, 28 H,
ph-CH2), 7.15 (brs, 70 H, ph-H). ESI MS (m=z): calcd. for 1481.0
([MþH]þ=2e); obsd. 1481.6.
General Procedure for the Synthesis of the Dendron 5
To the solution of trifluoroacetic acid (TFA)=anhydrous CH2Cl2 (1:1,
v=v), 4 was added at room temperature under N2. When the protecting
groups were completely removed, the solvent was rotary-evaporated.
The resulting product was dissolved in chloroform and basified with satu-
rated NaHCO3 to completely remove TFA. The organic layer was dried
over anhydrous Na2SO4, concentrated, and further purified on a

Synthesis of Peptide Dendrimers
4047
silica-gel chromatography column using CH2Cl2 and CH3OH (20:1, v=v)
as an eluent to afford 5.
General Procedure for the Synthesis of the Dendron Ia–c
The naproxen linker acid 2 (1.0 equiv) was added in one portion to a mix-
ture of dendron 5 (2.0 equiv), EDCIꢂ HCl (2.2 equiv), and Hobt (2.2equiv)
in DMF, keeping the reaction under N2. The mixture was stirred at ambi-
ent temperature overnight. Water and ethyl acetate were added, and the
mixture was stirred for 10min. The mixture was allowed to settle, and
the lower aqueous phase was separated off and discarded. The organic
phase was washed with more water and then concentrated under a reduced
vaccum on a rotary evaporator to give the crude product. The crude pro-
duct was purified on a silica-gel chromatography column using CH2Cl2
and CH3OH (80:1–30:1, v=v) as an eluent.
General Procedure for the Synthesis of the Dendron IIa–c
A mixture of dendron I (1.0 equiv) and 10% Pd=C (0.1 equiv, 10 wt%) in
CH3OH was stirred at room temperature under a H2 atmosphere. After
16 h, the mixture was passed through a membrane filter to remove the
catalyst and then evaporated under reduced pressure.
Data
Compound IIa
White foam (0.12g, 55% total yield in two steps) from 2 (0.03 g,
0.08mmol).1H NMR (400MHz, D2O): 1.46 (d, 3 H, J ¼ 6.0 Hz, NapCH3),
2.53–2.74 (m, 12H, CH2CO ꢃ 6), 2.80–3.04 (brs, 32H, Asp-bCH2),
3.42–3.69 (m, 12H, N-CH2 ꢃ 6), 3.76–3.89 (m, 4 H, Gly-CH2 ꢃ 2),
3.8–3.96 (brs, 4 H, NapOCH3 þ CH), 4.60–4.95 (m, 16H, Asp-aCH),
7.37–7.85 (m, 6 H, Nap-phH). MALDI-TOF MS (m=z): calcd. for
2638.7 ([M þ Na]þ); obsd. 2639.2. Anal. calcd. for C100H127N21O62: C,
45.93; H, 4.89; N,11.25. Found: C, 46.11; H, 4.82; N, 11.17.
Compound IIb
White foam (0.11 g, 43% total yield in two steps) from 2 (0.03 g,
0.08 mmol). 1H NMR (400 MHz, D2O): 1.47 (d, 3 H, J ¼ 6.0 Hz,

4048
L. Ouyang et al.
NapCH3), 2.44–2.60 (m, 12 H, CH2CO ꢃ 6), 2.86–3.06 (brs, 40 H,
Asp-bCH2), 3.38–3.60 (m, 12H, N-CH2 ꢃ 6), 3.77–3.88 (m, 4 H, Gly-CH2
ꢃ 2), 3.82–4.00 (brs, 4 H, NapOCH3 þ CH), 4.60–4.99 (m, 20H, Asp-
aCH), 7.30–7.88 (m, 6 H, Nap-phH). MALDI-TOF MS (m=z): calcd.
for 3074.8 ([M þ H]þ), obsd. 3076.1. Anal. calcd. for C116H147N25O74: C,
45.30; H, 4.82; N,11.39. Found: C, 45.12; H, 4.91, N, 11.47
Compound IIc
White foam (0.14 g, 51% total yield in two steps) from 2 (0.03 g,
0.08 mmol).1H NMR (400 MHz, D2O): 1.47 (d, 3 H, J ¼ 6.0 Hz,
NapCH3), 2.50–2.79 (m, 12 H, CH2CO ꢃ 6), 2.90–3.09 (brs, 48 H,
Asp-bCH2), 3.33–3.56 (m, 12 H, N-CH2 ꢃ 6), 3.82–3.91 (m, 4 H,
Gly-CH2 ꢃ 2), 3.83–4.01 (brs, 4 H, NapOCH3 þ CH), 4.62–4.89 (m,
24 H, Asp-aCH), 7.25–7.87 (m, 6 H, Nap-phH). MALDI-TOF MS
(m=z): calcd. for 3534.9 ([M þ H]þ); obsd. 3534.9. Anal. calcd. for
C132H167N29O86: C, 44.84; H, 4.76; N,11.49. Found: C, 44.71; H, 4.88;
N, 11.52
General Procedure for the Synthesis of the Dendron 6
The same procedure as described for preparation of the dendron I was
used.
Data
Compound 6a
Yellow wax (0.25 g, 61%) from 3 (0.03 g, 0.01mmol). 1H NMR (400MHz,
CDCl3):1.40 (s, 9 H, Boc-H), 2.36–2.52 (m, 12H, CH2CO ꢃ 6), 2.69–2.94
(brs, 32H, Asp-bCH2), 3.48–3.63 (m, 12H, N-CH2 ꢃ 6), 3.71–3.92 (m,
6 H, Gly-CH2 ꢃ 3), 4.72–4.94 (m, 16H, Asp-aCH), 5.14 (brs, 40H,
ph-CH2), 7.25 (brs, 100 H, ph-H). MALDI-TOF MS (m=z): calcd. for
4381.7 ([M þ Na]þ); obsd. 4382.4.
Compound 6b
Yellow wax (0.33 g, 67%) from 3 (0.03 g, 0.01mmol). 1H NMR (400MHz,
CDCl3):1.41 (s, 9 H, Boc-H), 2.42–2.51 (m, 12H, CH2CO ꢃ 6), 2.59–3.01
(brs, 40H, Asp-bCH2), 3.52–3.60 (m, 12H, N-CH2 ꢃ 6), 3.66–3.87 (m,

Synthesis of Peptide Dendrimers
4049
6 H, Gly-CH2 ꢃ 3), 4.65–4.99 (m, 20H, Asp-aCH), 5.21 (brs, 48H,
ph-CH2), 7.15 (brs, 120 H, ph-H). MALDI-TOF MS (m=z): calcd. for
5180.9 ([M þ H]þ); obsd. 5182.2.
Compound 6c
Yellow wax (0.26 g, 45%) from 3 (0.03 g, 0.01mmol). 1H NMR (400MHz,
CDCl3):1.41 (s, 9 H, Boc-H), 2.43–2.56 (m, 12H, CH2CO ꢃ 6), 2.64–2.92
(brs, 48 H, Asp-bCH2), 3.56–3.67 (m, 12H, N-CH2 ꢃ 6), 3.75–3.92 (m,
6 H, Gly-CH2 ꢃ 3), 4.71–5.04 (m, 24H, Asp-aCH), 5.20 (brs, 56H,
ph-CH2), 7.14 (brs, 140 H, ph-H). MALDI-TOF MS (m=z): calcd. for
6002.2 ([M þ H]þ); obsd. 6003.7.
General Procedure for the Synthesis of the Dendron 7
The same procedure as described for preparation of the dendron 5 was
used.
General Procedure for the Synthesis of the Dendrons IIIa–c
The naproxen linker acid 2 (1.0 equiv), the dendron 7 (2.0 equiv),
o-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU)
(2.2 equiv), and Hobt (2.2 equiv) were dissolved in dry DMF under
N2. N,N-Diisopropylethylamine (DIPEA) (2.2 equiv) was then added,
and the reaction was stirred for 24h at room temperature. The solvent
was evaporated, and the slurry was partitioned between ethyl acetate and
water. The aqueous layer was further extracted with ethyl acetate, and the
combined organic layers were dried and evaporated to give a yellow
foam. The crude product was purified on a silica-gel chromatography
column using CH2Cl2 and CH3OH (60:1–20:1, v=v) as an eluent.
General Procedure for the Synthesis of the Dendrons IVa–c
A mixture of dendron III (1.0 equiv) and 10% Pd=C (0.1 equiv, 10 wt%)
in CH3OH was stirred at room temperature under an H2 atmosphere.
After 24 h, the mixture was passed through a membrane filter to remove
the catalyst and then evaporated under reduced pressure. After concen-
tration, the residue was triturated with pure ethyl ether to afford IV as
a white powder.

4050
L. Ouyang et al.
Data
Compound IVa
White powder (0.051g, 36% total yield in two steps) from 2 (0.01g,
0.03mmol). 1H NMR (400MHz, D2O): 1.46 (d, 3 H, J ¼ 6.0 Hz, NapCH3),
2.53–2.74 (brs, 28H, CH2CO ꢃ 14), 2.77–2.94 (brs, 64H, Asp-bCH2),
3.39–3.62 (brs, 28H, N-CH2 ꢃ 14), 3.81–3.83 (brs, 12H, Gly-CH2 ꢃ 6),
3.89–3.96 (s, 4 H, NapOCH3 þ CH), 4.63–4.96 (m, 32H, Asp-aCH),
7.32–7.85 (m, 6 H, Nap-phH). MALDI-TOF MS (m=z): calcd. for
5255.5 ([M þ H]þ); obsd. 5258.3. Anal. calcd. for C196H255N45O126: C,
44.78; H, 4.89; N,11.99. Found: C, 44.41; H, 4.81, N, 12.17.
Compound IVb
White powder (0.035 g, 21% total yield in two steps) from 2 (0.01 g,
0.03 mmol). 1H NMR (400 MHz, D2O): 1.45 (d, 3 H, J ¼ 6.0 Hz,
NapCH3), 2.57–2.73 (brs, 28 H, CH2CO ꢃ 14), 2.80–2.96 (brs, 80 H,
Asp-bCH2), 3.41–3.66 (brs, 28 H, N-CH2 ꢃ 14), 3.80–3.83 (brs, 12 H,
Gly-CH2 ꢃ 6), 3.89–3.95 (s, 4 H, NapOCH3 þ CH), 4.66–4.95 (m, 40 H,
Asp-aCH), 7.32–7.87 (m, 6 H, Nap-phH). MALDI-TOF MS (m=z):
calcd. for 6197.7 ([M þ Na]þ); obsd. 6198.3. Anal. calcd. for
C228H295N53O150: C, 44.33; H, 4.81; N, 12.02. Found: C, 44.14; H,
4.66; N, 12.19.
Compound IVc
White powder (0.055g, 29% total yield in two steps) from 2 (0.01g,
0.03mmol).1H NMR (400MHz, D2O): 1.45 (d, 3 H, J ¼ 6.0 Hz, NapCH3),
2.44–2.69 (brs, 28H, CH2CO ꢃ 14), 2.79–3.02 (brs, 96H, Asp-bCH2),
3.48–3.57 (brs, 28H, N-CH2 ꢃ 14), 3.81–3.84 (brs, 12H, Gly-CH2 ꢃ 6),
3.89–3.92 (s, 4 H, NapOCH3 þ CH), 4.54–4.99 (m, 48H, Asp-aCH),
7.32–7.87 (m, 6 H, Nap-phH). MALDI-TOF MS (m=z): calcd. for
7095.9 ([M þ H]þ); obsd. 7102.1. Anal. calcd. for C260H335N61O174: C,
43.99; H, 4.76; N,12.04. Found: C, 43.65; H, 4.56; N, 12.28.
Hydroxyapatite (HAP) Binding Study
Conjugate solutions in PBS (5 mL, concentration 1 mg=mL) were
incubated with HAP (25 mg) for 2 h at 37ꢁC under mild shaking. After
centrifugation, the concentration of nonbound conjugates was measured

Synthesis of Peptide Dendrimers
4051
by UV spectrophotometry in 273 nm (a characteristic absorption of
naproxen).
ACKNOWLEDGMENT
This work was supported by the National Natural Science Foundation of
China (Project 20472055).
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