Ultrasmall dual-modality silica nanoparticle drug conjugates: Design, synthesis, and characterization

Article abstract of DOI:10.1016/j.bmc.2015.09.050

The physicochemical design and synthesis of effective cancer-directed and particle-based nanotherapeutic imaging agents remains a challenging task. Of critical importance is the ability to demonstrate maximum delivery, retention, and treatment efficacy for platforms designed to deposit their cargo at sites of disease without attendant dose-limiting toxicity. In this work, we describe dual-modality nanoparticle drug conjugates (NDCs) which utilize protease sensitive linkers to attached drug compounds and imaging labels to a clinically translated class of ultrasmall silica nanoparticle (C′ dots). We describe the synthesis and characterization of these linker-drug constructs. Linkers incorporating dipeptide enzyme substrates are attached to analogs of a prototypical epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), through a cleavable amide bond or para-aminobenzyloxycarbonyl (PABC) group. These constructs are conjugated onto C′ dots leading to the desired NDCs. These NDCs exhibit fast and predictable release kinetics in the presence of model proteases, and are stable in various biological media. Finally, in vitro assays show NDCs to be highly active in reducing phosphorylated EGFR levels in H1650 cells, a human tumor-derived cell line. The data suggests that NDCs exhibit desirable properties that warrant further development toward oncological therapy.

Full text of DOI:10.1016/j.bmc.2015.09.050

Accepted Manuscript
Ultrasmall Dual-Modality Silica Nanoparticle Drug Conjugates: Design, Syn-
thesis, and Characterization
Barney Yoo, Kai Ma, Li Zhang, Andrew Burns, Sonia Sequeira, Ingo
Mellinghoff, Cameron Brennan, Ulrich Wiesner, Michelle S. Bradbury
PII:
S0968-0896(15)30075-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.050
BMC 12597
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 August 2015
23 September 2015
30 September 2015
Please cite this article as: Yoo, B., Ma, K., Zhang, L., Burns, A., Sequeira, S., Mellinghoff, I., Brennan, C., Wiesner,
U., Bradbury, M.S., Ultrasmall Dual-Modality Silica Nanoparticle Drug Conjugates: Design, Synthesis, and
Characterization, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.050
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Ultrasmall Dual-Modality Silica Nanoparticle Drug Conjugates:
Design, Synthesis, and Characterization
Barney Yoo,a.* Kai Ma,b Li Zhang,a Andrew Burns,c Sonia Sequeira,d Ingo Mellinghoff,e,f
Cameron Brennan,e,g Ulrich Wiesner,b,* and Michelle S. Bradburya,h,*
aDepartment of Radiology, Sloan Kettering Institute for Cancer Research, New York,
New York 10065
bDepartment of Materials Science & Engineering, Cornell University, Ithaca, New York 14853
cGE Global Research Center, Niskayuna, New York
dInvestigational Products Core, Sloan Kettering Institute for Cancer Research, New York,
New York, NY 10065
eHuman Oncology and Pathogenesis Program, Sloan Kettering Institute for Cancer Research,
New York, NY 10065
fDepartment of Neurology, Sloan Kettering Institute for Cancer Research, New York, NY 10065
gDepartment of Neurosurgery, Sloan Kettering Institute for Cancer Research, New York, NY
10065
hMolecular Pharmacology and Chemistry Program, Sloan Kettering Institute for Cancer
Research, New York, NY 10065
*To whom correspondence should be addressed.
Email: yoob@mskcc.org, ubw1@cornell.edu, bradburm@mskcc.org

Abstract
The physicochemical design and synthesis of effective cancer-directed and particle-based
nanotherapeutic imaging agents remains a challenging task. Of critical importance is the ability
to demonstrate maximum delivery, retention, and treatment efficacy for platforms designed to
deposit their cargo at sites of disease without attendant dose-limiting toxicity. In this work, we
describe dual-modality nanoparticle drug conjugates (NDCs) which utilize protease sensitive
linkers to attached drug compounds and imaging labels to a clinically translated class of
ultrasmall silica nanoparticle (C' dots). We describe the synthesis and characterization of these
linker-drug constructs. Linkers incorporating dipeptide enzyme substrates are attached to analogs
of a prototypical epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI),
through a cleavable amide bond or para-aminobenzyloxycarbonyl (PABC) group. These
constructs are conjugated onto C' dots leading to the desired NDCs. These NDCs exhibit fast and
predictable release kinetics in the presence of model proteases, and are stable in various
biological media. Finally, in vitro assays show NDCs to be highly active in reducing
phosphorylated EGFR levels in H1650 cells, a human tumor-derived cell line. The data suggests
that NDCs exhibit desirable properties that warrant further development towards oncological
therapy.
Key word: nanoparticle; drug delivery; drug conjugate; kinase inhibitor; cancer.

1. Introduction
Nanotherapeutic delivery vehicles tailored for drug selection and delivery to stratified
subpopulations of patients have become a prominent feature in the current and emerging
repertoire of cancer therapies.1-4 These are typically macro- or supra-molecular multicomponent
systems, ranging in size from 1-1,000 nm, that are either inherently therapeutic (i.e., no active
pharmaceutical ingredient) or function as carriers of therapeutics which have been directly
coupled.5 To date, liposomal nanoparticles and biologics comprise a large proportion of the
number of products used to treat a variety of cancer types, and these agents have either been
approved or are still in clinical development;2,6 a number of polymer-based particle formulations
are also currently being evaluated in early phase trials.1,4 It is rather surprising, therefore, that of
these available nanotherapeutic products, there is a paucity of products possessing the molecular
imaging capabilities needed to predict biological properties (i.e., clearance, biodistribution) in
individual patients for optimizing cancer therapy. We propose and demonstrate an alternative
class of nanotherapeutic, known as a nanoparticle drug conjugateTM (NDC),7 in which drug
compounds and contrast-producing labels are covalently attached through protease sensitive
linkers to ultrasmall (sub-10 nm diameter) nanoparticles to produce a controlled release, dual-
modality platform for positron emission tomography (PET) and optically-driven applications.8-11
This combination of size, molecular composition and chemistry (i.e. mode of drug release), and
endowed imaging capabilities may leverage the beneficial properties seen in other
nanotherapeutic products with the aim of overcoming key obstacles hampering traditional
formulations: narrow therapeutic indices, dose-limiting toxicities, and limited clinical utility.12
Desirable candidates for nanotherapeutic delivery systems share a common feature of
incorporating and releasing a drug compound in a controlled manner, which can favorably alter

drug bioavailability and pharmacokinetics, while minimizing off-target toxicities. Ideally, an
imaging label has been incorporated to assess their precise localization and retention at disease
sites. However the mechanisms by which these systems function differ significantly. For
example, antibody drug conjugates (ADCs) achieve lower drug toxicity primarily through active
targeting of tumor cells and conditional release of drug molecules.2,4 Upon binding a cell surface
antigen, active drug release occurs after cellular internalization and endosomal uptake. On the
other hand, liposomes and polymer-based drug delivery systems, which are typically much larger
assembled complexes (~20-150 nm diameters) passively loaded with a greater payload (~10,000
drug molecules for Doxil)13 have generally lacked targeting capabilities (BIND-014 is an
exception),4 relying primarily on the well-known enhanced permeability and retention (EPR)
effect for the successful delivery of nano-formulated drugs.14 While interstitial permeation of
liposomes may be poor due to their size, the free drug is released through various mechanisms
that are not entirely understood. Abraxane (~140 nm) relies on a different approach to enhance
the bioavailability of a hydrophobic compound. A specific formulation of albumin and drug
(paclitaxel) forms the initial complex, which is then estimated to disperse into smaller protein-
drug aggregates upon injection.2,4
In this work, we focus on the design, synthesis, characterization, and preliminary biological
evaluation of NDCs as a first-in-kind ultrasmall organic-inorganic hybrid drug delivery vehicle
with dual-modality (PET-optical) imaging capabilities. In vivo imaging will be presented in a
subsequent paper, similar to that seen in prior studies.11,15 NDCs are comprised of an ultrasmall
(~6 nm diameter) poly(ethylene glycol) coated (PEGylated) fluorescent core-shell silica
nanoparticle, referred to as C' dot, with drugs and radiolabels covalently attached to the surface.
In a sense, these particles represent a ‘hybrid’ drug delivery system that draw upon particular

features of the aforementioned nanomedicines; NDCs are macromolecular in size, similar to that
of proteins (i.e., biologics), yet incorporate design features of other particle-based materials, such
as a PEGylated outer layer that renders the surface neutrally charged. These properties, in turn,
facilitate bulk renal clearance while enabling evasion of the reticuloendothelial system. The drug
component is covalently attached to the PEGylated surface for stability, and drug release is
achieved through enzymatic hydrolysis. We anticipate that the combination of these attributes
may lead to distinctly favorable properties for this therapeutic platform, including improved
control of drug release rates, quantitative pharmacokinetic profiles, and lack of toxicity, as well
as enhanced target tissue delivery, retention, and distribution.
2. Results and Discussion
2.1. Design strategy
Previously, we have reported on the design and synthesis of ultrasmall nanoparticles, C
dots16,17 with a range of modular functionalities. These are fluorescent, organo-silica core - silica
shell particles that have diameters controllable down to the sub-10 nm range. The silica shell
surface of the dye-containing core is covalently modified with silyl-polyethylene glycol (PEG)
groups to enhance stability in aqueous and biologically relevant conditions. These particles have
been evaluated in vivo and exhibit excellent clearance properties owing largely to their sub-10
nm size and inert surface.17,18 Among the additional functionalities incorporated into C dots are
chemical sensing,19,20 non-optical (PET) image contrast and in vitro/in vivo targeting capabilities
(enabling their use in visualizing lymph nodes for surgical applications, and melanoma detection
in cancer11,15). In fact, these particles are the first organic-inorganic hybrid particles to have
reached clinical trials in patients for molecular cancer imaging using either PET (or optical)

methods.21 Cost-effective synthetic approaches in aqueous media have recently led to the latest
generation dots, now referred to as C' dots, that exhibit improved size control, enhanced covalent
near infrared (NIR) dye incorporation efficiencies, and higher fluorescence brightness.22 The
present work was conducted exclusively with this latest particle generation.
We hypothesize that therapeutic C' dots, also adapted for molecular cancer imaging, may
provide a unique platform for drug delivery owing to their physical properties, as well as
demonstrated human in vivo characteristics.21 In conceptualizing C' dot-based NDCs for drug
delivery, we sought to develop a general strategy that would provide good biostability (to
minimize premature drug release) and exhibit controlled release of the bioactive compound at the
desired site (Figure 1) – properties that could potentially maximize target tissue bioavailability
while abrogating dose-limiting toxicity. The addition of a radiolabel for PET imaging facilitates
tumor visualization, quantifies tumor accumulation, and permits tracking of either the drug or
particle in vivo. Studies with antibody and polymer drug conjugates suggest peptide-based
linkers to be ideal candidates for NDC applications.23-25 These linkers, in the context of
antibodies and polymers, have been shown to be stable both in vitro and in vivo, with highly
predictable release kinetics that rely on enzyme-catalyzed hydrolysis, presumably by lysosomal
proteases. For example, cathepsin B, a highly expressed protease in lysosomes, has been utilized
to facilitate drug release from macromolecules.26
Enzyme cleavage site
In cells
Drug
Protease mediated drug release
Peptide
Peptide
Drug
C’ dot
Cleavable linker-drug
construct
C’ dot
Figure 1. Schematic of enzyme-mediated drug release from nanoparticle drug conjugate (NDC). Blue dots represent fluorescent
PEGylated core-shell silica nanoparticles.

The model drug selected for this work is gefitinib – a receptor tyrosine kinase inhibitor
(TKI) to the epidermal growth factor receptor (EGFR).27 EGFR has emerged as an attractive
molecular candidate for targeted therapy.28 EGFR mutations resulting in constitutive activation
have been found in 25% of metastatic non-small cell lung cancer (NSCLC) and 40-50% of
primary glioblastoma multiforme (GBM) – two prevalent forms of brain cancer.29 While EGFR-
TKIs have shown promise in preclinical studies, they have been shown to be largely ineffective
in malignant brain cancer patients. This is likely due to their poor pharmacokinetic profiles and
tissue or central nervous system (CNS) penetration, as well as dose-limiting toxicity,12,30,31 i.e. all
factors that may be potentially improved via use of an appropriate NDC.
2.2. Synthesis of gefitinib analogues
Gefitinib is designed to bind and inhibit the kinase domain active site of EGFR.32 To utilize
the anilinoquinazoline component of gefitinib in the context of NDCs, it was necessary to
incorporate a chemically reactive group that would not significantly perturb drug binding to the
kinase domain. From reported X-ray crystallographic and structure-active relationship (SAR)
studies,27,32 we hypothesized that replacing the terminal heterocycle (morpholino) group with an
amine, as shown in Figure 2a, would not significantly alter drug activity but would provide the
needed chemical functionality (amine) for modification and eventual covalent attachment to the
C' dot. With the commercial availability of 6, des-morpholino-gefitinib (dMG), we readily
obtained the desired APdMG (1) through a one-step nucleophilic substitution of Boc protected
amino propyl bromide, followed by acid deprotection (Scheme 1).

Figure 2. Chemical structure of (a) gefitinib and gefitinib analogues (APdMG and dPEG2APdMG); (b) linker-drug directly
connected through amide bond (3); (c) linker-drug connected through dPEG2 spacer (4); (d) linker-drug connected through
degradable PABC spacer (5).
Scheme 1. a) K2CO3, DMF, 60°C; b) TFA/H2O (9:1); c) HATU, DIEA, DMF; b) piperidine, DMF.

Additionally, the gefitinib analogue dPEG2APdMG (2), which contains an extended 10 atom
PEG spacer, was prepared. This analogue was needed to address potential steric problems a
protease, for example, might encounter due to the close proximity of the peptide linker and drug.
Compound 2 was readily obtained by reacting 1 with Fmoc-dPEG2-COOH using standard
coupling methods. The isolated product was then deprotected with piperidine/DMF (Scheme 1).
H1650 cells were treated with these compounds and analyzed by western blot to assess phospho-
Tyr168 levels in EGFR to ensure that in particular 1 retained activity against EGFR. The H1650
cells are a model human tumor-derived non-small-cell lung cancer (NSCLC) line
(bronchioalveolar carcinoma), which contain a mutated EGFR (L858R and ΔE746-A750)
resulting in constitutive activity of the receptor. Indeed, compound 1 showed effects similar to
gefitinib, with inhibition of phospho-Tyr168 at 1 and 10 μM concentrations, and compound 2
showed reduced activity (data not shown).
2.3. Synthesis of linker-drug constructs
Three linker-drug designs were investigated for C' dot-based drug delivery in this study
(Figure 2b-d). Inspired by previously reported work with antibody drug conjugates,26 both linker
types revolve around dipeptide sequences which utilize proteases for drug release. Proteases
recognize and bind the dipeptide leading to hydrolysis at the C-terminal end, releasing the drug
component from the linker. Two model proteases were used to evaluate the linker-drug
constructs described here, trypsin and cathepsin B. Trypsin was selected as a representative
serine protease. It is highly active against peptides containing basic amino acids such as arginine
and lysine, and cleaves C-terminals to these residues. Cathepsin B is a cysteine protease with
more stringent substrate specificity. The minimal substrate consensus sequence described to date
is a dipeptide motif containing a hydrophobic and basic residue.26,33 Similar to trypsin, cathepsin

B cleaves C-terminals to the basic amino acids. Thus the dipeptides phenylalanine-arginine (Phe-
Arg) and phenylalanine-lysine (Phe-Lys), both recognition motifs for trypsin and cathepsin B,
have been incorporated into the linker-drug constructs (Figure 3).
Figure 3. Three linker-drug constructs. (a) Phe-Arg-APdMG utilizes an amide bond for drug attachment. Enzymes recognize and
bind the dipeptide sequence (Phe-Arg) then hydrolyze the amide bond C-terminal to the dipeptide and release APdMG, 1; (b) to
enhance drug release, Phe-Arg-dPEG2APdMG uses 2, which incorporates a longer 10 atom PEG spacer between the drug and
dipeptide; (c) Phe-Lys-PABC-APdMG utilizes a para-aminobenzoxy-carbamate (or PABC) spacer group between the dipeptide
(Phe-Lys) and aminopropyl-dMG. After enzyme catalyzed release of the spacer-drug, the spacer spontaneously decomposes from
the drug.
The Phe-Arg-APdMG (3) represents the most straightforward approach to obtaining a
protease sensitive linker-drug construct (Figure 2b, 3a). In this design, the gefitinib analog 1 is
directly attached to the C-terminus of the dipeptide sequence. Compound 3 was obtained using
solid phase peptide synthesis methods leading to 9; followed by coupling of the C-terminus with
2 leading to 10 and a final acid deprotection step to yield 12 (Scheme 2). The N-terminal amine
was then reacted with SAMA-OPfp; 14 is then treated with base resulting in the desired product
3. Compound 4 was obtained in a similar manner (Scheme 2), except the gefitinib analog 2 was

coupled with 9 resulting in the precursor 11. Deprotection of 11 yielded 13, where the N-
terminus was then converted into the S-acetyl acetamide (15). The final treatment with NaOH
afforded Phe-Arg-dPEG2APdMG (4) – a linker-drug construct containing a 10 atoms PEG spacer
between the Phe-Arg and the gefitinib analog (Figure 2c, 3b).
Scheme 2. a) SPPS; b) HFIP in DCM; c) HATU, DIEA, DMF; d) TFA; e) DIEA, DMF; f) NaOH
The increased spacing between the dipeptide and drug components without introducing
structural changes to 2 were retained by designing and synthesizing a third construct, Phe-Lys-
PABC-APdMG (5). This linker incorporates a so-called self-immolative34 para-aminobenzyloxy
carbonyl (PABC) group between the peptide and drug (Figure 2d, 3c): Upon enzymatic
hydrolysis, this group further decomposes into para-aminobenzyl alcohol (PABA) and CO2,
thereby releasing the drug compound APdMG. Synthesis of compound 5 begins with Fmoc-
Lys(Mtt)-OH (Scheme 3).

Scheme 3. a) para-Aminobenyl alcohol, HATU, DIEA, DMF; b) 30% piperidine in DMF; c) Fmoc-Phe, HATU, DIEA, DMF; d)
para-nitrophenyl chloroformate, pyridine, DCM; e) DIEA, DMF; f) 30% piperidine in DMF; g) Mtt-S-dPEG8-COOH, HATU,
DIEA, DMF; h) 0.5% TFA, 5% TIS in DCM.
The C-terminus of the protected amino acid is modified with para-aminobenzyl alcohol giving
Fmoc-Lys(Mtt)-PABA (16). Upon removal of the Fmoc group with piperidine, and coupling
with Fmoc-Phe-OH, the protected dipeptide Fmoc-Phe-Lys(Mtt)-PABA (17) is obtained. The
free hydroxyl group of the terminal PABA is then reacted with para-nitrophenol carbonate
chloride leading to an activated carbonate (18), which is further reacted with 1 resulting in
compound 19. The Fmoc group is removed, and the free N-terminal amine is coupled with Mtt-
S-dPEG8-COOH, yielding 20. The final deprotection step required acidic conditions; however,
the para-aminobenzyloxy carbamate group itself is susceptible to decomposition under such

conditions. A sufficiently mild condition (0.5% TFA) was found so as to remove the Mtt group
from the lysine and Mmt group from the terminal thiol while preserving the linker to afford the
desired product 5. It should be noted that the Mtt group masking the lysine side chain proved to
be ideally suited for this overall synthetic approach as it was found to be stable in the presence of
para-nitrophenol carbonate chloride, but sufficiently labile for removal under mild acidic
conditions. This is in contrast to the more commonly used hyper-labile Mmt group for lysine side
chain protection,35 that can be readily removed in the presence of para-nitrophenol carbonate
chloride.
2.4. Enzymatic evaluation of linker-drug constructs
In vitro assays were conducted for compounds 3 to 5 to evaluate drug release profiles;
enzyme mediated hydrolysis of the constructs was monitored over different time points (Table 1,
Figure S1 and S2 in Supplementary Material). For both trypsin and cathepsin B, the rate of drug
release for the three linker-drug constructs follows the same general trend: 5 > 4 > 3 (fastest to
slowest).
Table 1. Drug Release Assay for Linker-Drug Constructs
Trypsin
Cathepsin B
Substrate
t1/2 a (min)
t1/2 a (min)
Phe-Arg-APdMG (3)
9
2
1
NH
110
1
Phe-Arg-dPEG2APdMG (4)
Phe-Lys-PABC-APdMG (5)
NH – no hydrolysis
a
Time when 50% of the drug is released from linker or particle as determined
by HPLC at 348 nm.
The results for 3 and 4 suggest that the proximity of the drug to the dipeptide unit affects enzyme
activity and drug release. Hydrolysis (drug release) is enhanced when the spacing (distance)

between the drug and dipeptide is increased through the incorporation of a 10 atom PEG linker.
This effect is most prominently observed with cathepsin B, which is unable to hydrolyze
construct 3. However, by incorporating the 10 atom PEG spacer between the drug and dipeptide
(4), hydrolysis and drug release are observed.
2.5. Synthesis and characterization of NDCs
In order to obtain NDCs, maleimide-functionalized C' dots (mal-PEG-Cy5-C' dots) were
first synthesized, as reported in detail elsewhere.22 The NDCs, C' dot-(Cy5)-PEG-Phe-Arg-
dPEG2APdMG (6) and C' dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG (7) were prepared by adding
Phe-Arg-dPEG2APdMG (4) and Phe-Lys-PABC-APdMG (5) to the mal-PEG-C' dots to allow
the terminal thiols on the constructs to react with maleimide groups on the particle surface
(Scheme 4).
Scheme 4. C' dots-(Cy5)-PEG-mal are reacted with linker-drug constructs Phe-Arg-dPEG2APdMG (4) and Phe-Lys-PABC-
APdMG (5) resulting in the NDCs 6 and 7.
The naming convention used for the NDCs was adopted to reflect the chemical directionality and
connectivity of the linker-drug constructs. Consequently, the name of the linker-drug is placed
after the C' dot, as opposed to before, the latter convention adopted in our previous nomenclature
(e.g. cRGD-Cy5-C' dot). The desired NDC products 6 and 7 were isolated following purification
by gel filtration (Figure S3). All final particle products (C' dots and NDCs) were studied by

analytical reversed phase HPLC, transmission electron microscopy (TEM), optical
absorption/emission spectroscopy and fluorescence correlation spectroscopy (FCS) in order to
characterize overall purity, morphology, size, fluorescence properties. The NDCs 6 and 7 were
prepared with good overall purity and uniformity as determined by HPLC at 348 nm and TEM
(Figure 4a and 4b).
(a)
(b)
NDC (6)
NDC (7)
2
4
6
8
10
Time (min)
(c)
(d)
Figure 4. Characterization of mal-PEG-C' dots and NDC 6 and 7. (a) Analytical C18 reversed phase HPLC at 348 nm; (b) TEM;
(c) absorbance and emission spectra; (d) FCS correlation curves.
Absorption and emission spectra of optical density matched (around 650 nm) samples of Cy5
free dye, mal-PEG- C' dots, and NDCs 6 and 7 were obtained (Figure 4c). The absorption spectra
reveal a peak at around 340 nm, in addition to the Cy5 peak at around 650 nm, for the NDCs,
which matches well with the absorbance of gefitinib and suggests successful attachment of the
drug-linker constructs onto the PEGylated C' dot surface. According to the emission spectra, as

expected maleimide functionalized C' dots and NDCs show the same brightness enhancement
relative to free Cy5 dye of around 1.4 confirming that outer surface functionalization does not
change the particle fluorescence properties. Finally, FCS measurements suggest that particle size
only slightly increases after conjugation with drug-linker constructs. In Figure 4d FCS auto-
correlation curves shift to larger times, t, implying slower diffusive motion as a result of the
larger particle size. From fits of the auto-correlation curves also shown in Figure 4d (inset), the
hydrodynamic size of the particles (Table 2) only slightly increases from originally 6.3 nm (C'
dot) to 6.4 nm (NDC 6) and 6.5 nm (NDC 7). Considering that the fully extended drug-linker
constructs are at least 2 nm in length, this slight increase of particle size further implies that the
actual configuration of the drug-linker constructs on the particle surface most likely is not
extended, but rather folded.
Table 2. Nanoparticle Characterization Summary
Diameter a
DPR
Particle
Average b
(nm)
C’dot-(Cy5)-PEG-Mal
6.3
6.4
6.5
−
5
2
C’dot-(Cy5)-PEG-Phe-Arg-dPEG2APdMG (6)
C’dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG (7)
DPR – drug to particle ratio
a Determined by FCS
b Determined by HPLC
Analytical HPLC was also used to assess the number of drug molecules per particle or
drug to particle ratio (DPR) (Table 2). The concentration of gefitinib analogs can be readily
measured at 348 nm, while particle concentrations can be obtained at 650 nm, due to the Cy5
embedded within the C' dots. For the NDCs used in these studies, the average DPRs were modest,
5 and 2 for NDC 6 and 7, respectively. More recently DPRs in the range of 10 have been

obtained and will be utilized in upcoming studies. It is worth noting that despite the poor
solubility of the gefitinib analogues precipitation of NDCs was not observed, most likely due to
the high dispersibility of C' dots.
2.6. In vitro evaluation of NDCs
Enzyme dependent drug release over time was measured for C' dot-(Cy5)-PEG-Phe-Arg-
dPEG2APdMG 6 and C' dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG 7 to obtain in vitro drug
release profiles (Figure 5). Representative HPLC data demonstrating drug release with trypsin is
shown in Figure 6. NDCs 6 and 7 proved to be excellent substrates for trypsin, requiring 44 min
and 6 min, respectively, to achieve 50% drug release (Figure 5a, Table 3). Surprisingly, in the
presence of cathepsin B, release kinetics were markedly slower for both NDCs: 50% of drug
release was achieved in 560 min for 6 and 510 min for 7 (Figure 5b, Table 3). Taken together,
the data demonstrates accessibility of the linker-drug constructs on the particle surface to
enzymes, leading to controlled release of the drug components.
(a)
(b)
NDC (Cathepsin B)
NDC (Trypsin)
100
50
0
100
50
0
0
500
1000
1500
0
50
100
150
200
Time (min)
Time (min)
C'dot-(Cy5)-PEG-Phe-Arg-dPEG2APdMG
C'dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG
Figure 5. In vitro drug release from NDCs 6 and 7 in the presence of enzymes. C' dot-(Cy5)-PEG-Phe-Arg-dPEG2APdMG 6
and C' dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG 7 were treated with either trypsin (a) or cathepsin B (b). Enzymatic reactions
were monitored over time by HPLC at 348 nm. Trypsin assays were conducted in 10 mM phosphate buffer (pH 7.2) at 37°C;
cathepsin B assays were conducted in 25 mM sodium acetate buffer (pH 5.0) at 37°C.

2
1
(a)
(b)
6
7
120 min
5 min
120 min
5 min
Time (min)
Time (min)
C' dot-(Cy5)-PEG-Phe-Arg-dPEG2APdMG (6)
C' dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG (7)
+ Trypsin
+ Trypsin
Figure 6. Representative HPLC profiles of in vitro drug release from NDCs 6 and 7 in the presence of trypsin. NDCs were
treated with trypsin then analyzed by HPLC after 5 and 120 min. (a) NDC 6; (b) NDC 7. Data indicate that compounds 2 or 1 are
released from the C’dots. Trypsin assays were conducted in 10 mM phosphate buffer (pH 7.2) at 37°C. HPLC analysis was
performed at 348 nm.
Table 3. Drug Release Assay for NDCs
Trypsin
Cathepsin B
Substrate
t1/2 a (min)
t1/2 a (min)
C’dot-(Cy5)-PEG-Phe-Arg-dPEG2APdMG (6)
C’dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG (7)
44
6
560
510
a Time when 50% of the drug is released from linker or particle. Determined by HPLC.
The stabilities of NDCs were evaluated in aqueous solutions under acidic and neutral pH
conditions (5.0 and 7.2) at 37ºC. Both NDCs exhibited minimal degradation or drug release after
48 hours, as assessed by HPLC (Table 4). Recently, thiol-maleimide based conjugations have
drawn scrutiny due to the observed loss of linker drug constructs from antibody drug conjugates
due to possible reverse Michael or exchange reactions that can occur with free thiols. To assess
in vitro stability of the NDCs in the presence of excess thiols, 7 was incubated with 30 mM

gluthathione at 37ºC for 48 hours at pH 7.2. Less than 5% of the linker-drug was separated from
the C' dot after 48 hours (Table 4).
Table 4. NDC Stability Data
pH 5.2 a
48 hr
pH 7.2 b
48 hr
Glutathionec
48 hr
Mediad
18 hr
Particle
NDC 6
NDC 7
~ 1%
~ 6%
~ 2%
~ 2%
~ 5%
~ 4%
< 5%
< 1%
a 25 mM sodium acetate buffer
b 50 mM phosphate buffer
c 10 mM glutathione (reduced) in 50 mM phosphate buffer
d DEM, serum free, 18 hrs after cell treatment
The biological activities of C' dot-(Cy5)-PEG-Phe-Arg-dPEG2APdMG 6 and C' dot-
(Cy5)-PEG-Phe-Lys-PABC-APdMG 7 were assessed by treatment of H1650 cells followed by
western blot detection of phospho-Tyr168 in EGFR and compared with gefitinib. Serum starved
cells were incubated with each NDC over a period of 18 hours, and then subjected to EGF
stimulation. As expected, the gefitinib control exhibited a dose-dependent decrease in Tyr168
phosphorylation of EGFR, with complete inhibition at 1 μM (Figure 7).
Figure 7. Western blot analysis of H1650 cells treated with gefitinib, C' dot-(Cy5)-PEG-Phe-Arg-dPEG2APdMG (NDC 6), and
C' dot-(Cy5)-PEG-Phe-Lys-PABC-APdMG (NDC 7). Cells treated with gefitinib or specified NDC at indicated concentrations
for 18 hrs followed by EGF (50 ng/mL) for 5 minutes. (pEGFR – phosphorylated EGFR; tEGFR – total EGFR)

NDCs 6 and 7 also showed dose-dependent inhibition (Figure 7). However, cells treated
with 6 showed detectable levels of phospho-Tyr168 at 10 μM. By contrast, compound 7, which
had a lower DPR (Table 2), exhibited increased potency relative to 6 and the gefitinib control; a
marked loss of phospho-Tyr168 expression was observed at 100 nM of compound 7, with
complete inhibition at NDC concentrations of 1 μM. Thus, even though NDC 7 had a lower
DPR, it incorporated a more active gefitinib analogue 3, as against analogue 2 used in NDC 6.
Given the possibility that premature drug release can occur and lead to the observed decreases in
phospho-Tyr168EGFR, we monitored the stability of the NDCs used in these assays. An aliquot
of the media with NDC 6 or 7 (10 μM) used for treating the H1650 cells for 18 hours was
analyzed by HPLC. Both particles proved to be stable under these conditions as no free drug was
detected in the media and NDCs were intact (Table 4).
2.7. Radiolabeled NDCs
In addition to the NDCs 6 and 7, we investigated the potential for incorporating a
secondary imaging modality for future in vivo studies. To this end, we synthesized a linker-drug
construct, Phe-Arg-dPEG2-D-Tyr-aminopropyl-dMG that incorporates a D-tyrosine residue with
the drug component for attaching a radiolabel (compounds 21 and 23 in Supplementary Material).
The NDC C' dot-(Cy5)-PEG-Phe-Arg-dPEG2-D-Tyr-APdMG was prepared, and successfully
radioiodinated with 131I at >90% radiochemical purity (Figure S4).
3. Conclusion
In this report we introduce nanoparticle drug conjugates composed of a clinically
translated silica-based nanoparticle platform with covalently attached drug molecules and
contrast-producing radiolabels. The NDCs are ultrasmall (6-7 nm) and utilize enzyme sensitive
linkers, where drug release is catalyzed by proteases. Analogs of gefitinib, an important EGFR-

TKI cancer drug, was modified with these linkers and conjugated onto the PEGylated particle
surface. The resulting NDCs exhibited excellent in vitro stability, solubility, and proved to be
active in NSCLC cells, demonstrating improved potency at 100 nM concentrations over the
native drug itself. The attachment of a radiolabel will enable accurate quantification of NDC
biological properties for optimizing cancer therapies and improving patient selection on the basis
of observing successful particle localization at the tumor site. Investigations into the effects of
drug loading, the use of additional targeting surface ligands, and in vivo studies (stability and
pharmacokinetics) using relevant brain tumor mouse models are underway.
4. Experimental section
4.1. General information
Gel permeation chromatography (GPC) characterization was performed using a BioLogic LP
system equipped with a 275nm UV detector and with resin Superdex 200 from GE healthcare.
Transmission electron microscopy (TEM) images were taken using a FEI Tecnai T12 Spirit
microscope operated at an acceleration voltage of 120kV. Absorbance and fluorescence spectra
were measured by a Varian Cary 5000 spectrophotometer or Photon Technologies International
Quantamaster spectrofluorometer. FCS measurements were conducted using a home built FCS
setup with a 633nm solid-state laser as the laser source for Cy5. Silica gel purifications were
conducted on a Teledyne ISCO CombiFlash Rf. Analytical HPLC were run on a Waters Alliance
HPLC System or Autopure LCMS System (2767 Sample Manager, 2996 Photodiode Array
Detector, 2420 ELS Detector, Micromass ZQ, 2525 Binary Gradient Module, Column Fluidics
Organizer, 515 HPLC Pump, Pump Control Module II). HPLC purifications were performed on
a Waters Preparative System (2996 Photodiode Array Detector, 2545 Binary Gradient Module)

1
13
or Autopure LCMS System. H-NMR and C-NMR data were obtained on a Bruker Ultrashield
500 Plus. Further instrument details are included in Supplemental Materials.
4.2. Materials
Solvents and reagents purchased from Sigma Aldrich and Fisher Scientific unless otherwise
noted. Maleimide functionalized Cy5 fluorophores were purchased from GE healthcare. Silanes
were obtained from Gelest. PEGylated reagents were purchased from Quanta BioDesign. O-Des-
morpholinopropyl gefitinib was obtained from Toronto Research Chemicals (TRC). Amino acids
and peptide synthesis reagents were purchased from EMD Chemicals. DMSO-d and CDCl3 were
purchased from Cambridge Isotopes. Silica TLC plates, 4 g, 12 g, 24 g, and 40 g RediSep Rf
normal phase cartridges were obtained from Teledyne ISCO.
4.3 Synthesis
4.3.1. O-des-Morpholino-gefitinib, dMG (6)
1
Compound was obtained commercially. H-NMR (500 MHz, DMSO-d6): δ 9.69 (s, 1H), 9.47 (s,
1H), 8.48 (s, 1H), 8.22 (dd, J = 6.9, 2.7 Hz, 1H), 7.84 (ddd, J = 9.1, 4.4, 2.7 Hz, 1H), 7.78 (s, 1H),
13
7.41 (t, J = 9.1 Hz, 1H), 7.22 (s, 1H), 3.98 (s, 3H). C-NMR (125 MHz, DMSO-d6): δ 155.83,
153.91, 151.89, 146.73, 146.20, 122.74, 121.69, 116.50, 116.33, 109.51, 107.18, 105.25, 55.92.
ESI-MS (m/z) for C15H11ClFN3O2 (exact mass 319.1): [M+H]+ calc. 320.1, obs. 320.2.
4.3.2. N-(tert-Butyloxycarbonyl)-aminopropyl-dMG (7)
500 mg of 6 was dissolved in 100 mL of anhydrous DMF. K2CO3 (650 mg, 4.7 mmol, 3 eq) was
added to the solution. Stir bar. The reaction proceeded at 60°C for 16 hrs and checked by TLC
and/or HPLC. The solvent was removed in vacuo leaving a brown oil, which was flash purified
on a 24 g RediSep Rf normal phase cartridge using a linear gradient of DCM to 20% MeOH in
1
DCM. The final product was isolated as a white solid (312 mg, 62% yield). H-NMR (500 MHz,

DMSO-d6): δ 9.57 (s, 1H), 8.49 (s, 1H), 8.12 (dd, J = 6.8, 2.7 Hz, 1H), 7.81 (m, 1H), 7.44 (t, J =
9.1 Hz, 1H), 7.20 (s, 1H), 6.92 (t, J = 5.7 Hz, 1H), 4.17 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H), 3.15 (q,
J = 6.5 Hz, 2H), 1.95 (p, J = 6.5 Hz, 2H), 1.38 (s, 9H). 13C-NMR (125 MHz, DMSO-d6): δ
156.01, 155.59, 154.39, 152.61, 146.92, 123.40, 122.29, 118.62, 116.54, 116.37, 107.23, 102.59,
77.51, 66.69, 55.84, 37.22, 35.75, 28.95, 28.22. ESI-MS (m/z) for C23H26ClFN4O4 (exact mass
476.2): [M+H]+ calc. 477.2, obs. 477.3.
4.3.3. Aminopropyl-O-des-morpholino-gefitinib, APdMG (1)
7 (100 mg, 0.21 mmol) was treated with 1 mL TFA:water (9:1) for 30 min. The TFA:water was
removed in vacuo, leaving a light yellow oil. The oil was washed with diethyl ether, then
dissolved in a solution of water:acetonitrile (1:1), frozen and lyophilized. A tan solid was
obtained (TFA salt, 98 mg, 95% yield). 1H-NMR (500 MHz, DMSO-d6): δ 10.73 (s, 1H), 8.80 (s,
1H), 8.03 (s, 1H), 8.05 – 8.00 (m, 1H), 7.88 (s, 3H), 7.72 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.54 (t,
J = 9.0 Hz, 1H), 7.35 (s, 1H), 4.27 (t, J = 5.9 Hz, 2H), 4.00 (s, 3H), 3.04 (p, J = 6.7, 6.3 Hz, 2H),
2.13 (dt, J = 12.2, 6.0 Hz, 2H). 13C-NMR (125 MHz, DMSO-d6): δ 158.02, 148.80, 116.96,
116.79, 107.60, 103.66, 66.17, 56.42, 36.39, 26.59. ESI-MS (m/z) for C18H18ClFN4O2 (exact
mass 376.1): [M+H]+ calc. 377.1, obs. 377.2.
4.3.4. 9-Fluorenylmethoxycarbonyl-N-amido-dPEG2-aminopropyl-dMG, Fmoc-
dPEG2APdMG (8)
A solution containing 1 (25 mg, 0.05 mmol, TFA salt) and Fmoc-N-amido-dPEG2-COOH (20
mg, 0.05 mmol) in DMF (500 μL) was prepared. DIEA (19 mg, 0.15 mmol, 26 μL) was added
followed by a solution of HATU (19 mg, 0.05 mmol) in DMF (100 μL). The reaction proceeded
for 30 min at room temp, and determined complete by LCMS. The volume was reduced in vacuo,
and purified by silica gel chromatography using a gradient of ethyl acetate and 10% methanol in

ethyl acetate. Fractions were collected, pooled and solvent removed in vacuo. The isolated
1
product was a white solid (84% yield). H-NMR (500 MHz, DMSO-d6): δ 9.55 (d, J = 10.7 Hz,
1H), 8.51 (d, J = 2.4 Hz, 1H), 8.18 – 8.07 (m, 1H), 7.96 (t, J = 5.5 Hz, 1H), 7.87 (d, J = 7.6 Hz,
2H), 7.83 – 7.76 (m, 2H), 7.67 (d, J = 7.5 Hz, 2H), 7.49 – 7.36 (m, 3H), 7.31 (td, J = 7.5, 1.2 Hz,
3H), 7.21 (s, 1H), 4.28 (d, J = 6.9 Hz, 2H), 4.24 – 4.10 (m, 3H), 3.94 (s, 3H), 3.60 (t, J = 6.4 Hz,
2H), 3.46 (s, 4H), 3.36 (t, J = 6.0 Hz, 2H), 3.27 (q, J = 6.6 Hz, 2H), 3.10 (q, J = 5.9 Hz, 2H),
2.32 (t, J = 6.5 Hz, 2H), 1.97 (p, J = 6.5 Hz, 2H). 13C-NMR (125 MHz, CDCl3): δ 170.08, 143.85,
127.54, 126.98, 125.10, 120.05, 102.60, 69.42, 69.04, 66.78, 66.58, 55.87, 46.69, 40.01, 39.94,
39.85, 39.77, 39.68, 39.60, 39.51, 39.43, 39.35, 39.25, 39.18, 39.07, 39.01, 36.16, 35.69, 28.67,
0.08. ESI-MS (m/z) for C40H41ClFN5O7 (exact mass 757.27): [M+H]+ calc. 758.3, obs. 758.4.
4.3.5. Amino-dPEG2-aminopropyl-dMG, dPEG2APdMG (2)
8 (10 mg, 0.013 mmol) was dissolved in 30% piperidine in DMF (1 mL) and allowed to react for
15 min at room temp. The solvent was removed in vacuo, dissolved in water/acetonitrile and
purified by reversed phase (C18) HPLC. The product was recovered as a white powder (7 mg,
80% yield). 1H-NMR (500 MHz, DMSO-d6): δ 10.66 (s, 1H), 8.78 (s, 1H), 8.05 – 7.97 (m, 3H),
7.80 (s, 3H), 7.72 (ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.54 (t, J = 9.1 Hz, 1H), 7.32 (s, 1H), 4.20 (t, J
= 6.1 Hz, 2H), 4.00 (s, 3H), 3.65 – 3.48 (m, 7H), 3.27 (q, J = 6.6 Hz, 2H), 2.96 (q, J = 5.5 Hz,
2H), 2.34 (t, J = 6.5 Hz, 2H), 1.98 (t, J = 6.5 Hz, 2H). 13C-NMR (125 MHz, DMSO-d6): δ
116.96, 69.55, 69.29, 66.73, 66.61, 56.38, 38.57, 36.03, 35.63, 28.58. ESI-MS (m/z) for
C25H31ClFN5O5 (exact mass 535.20): [M+H]+ calc. 536.2, obs. 536.3.
4.3.6. Boc-N-amido-(dPEG2)3-Phe-Arg(Pbf)-OH (9)
Chlorotrityl resin (100 mg, 0.1 mmol, 1 mmol/g) was transferred into a fritted syringe reaction
vessel and suspended in 2 mL anhydrous DCM for 10 min. The solvent was dispensed, and a

solution of DIEA in anhydrous DCM followed by a solution of Fmoc-Arg(Pbf)-OH (97.5 mg,
1.5 eq) in anhydrous DCM were drawn into the syringe; and agitated for 40 min. The solution
was dispensed and the resin washed for 2 min, 2x with DCM, then 2x with DMF. Standard solid
phase peptide synthesis procedures were the carried out to obtain the final peptide. In short Fmoc
deprotections were accomplished by washing the resin 2x using 30% piperidine/DMF (1 mL) for
10 min. This was followed by DMF (1 mL) washes, 4x for 2 min each. Coupling reactions were
carried out at room temp using an excess of protected amino acid (3 eq in 2 mL DMF), DIEA
(120 mg, 0.9 mmol, 160 μL, 9 eq in 1 mL DMF), HATU (114 mg, 0.3 mmol, 3 eq in 2 mL
DMF), added to the syringe in that order, and shaken for 1 hr. This was followed by DMF (1
mL) washes, 4x for 2 min each. Fmoc-Phe (116 mg, 0.3 mmol, 3 eq) was added, followed by
three residues of Fmoc-N-dPEG2-OH (120 mg, 0.3 mmol, 3 eq). After completion of the
sequence, final Fmoc deprotection and washes, BOC anhydride (87 mg, 0.4 mmol, 4 eq) and
DIEA (120 mg, 0.9 mmol, 160 μL, 9 eq) in 2 mL of DMF were used to cap the N-terminal amine.
The peptide-resin was washed with DMF (1 mL, 2 min, 2x), then DCM (1 mL, 2 min, 4x). The
protected peptide product was then cleaved off of the resin by adding 50% HFIP in DCM (2 mL)
to the syringe and shaking for 1 hr at room temp. The crude peptide was then purified by
reversed phase (C18) HPLC, lyophilized, resulting in a white solid (62 mg). Analytical HPLC:
Figure S5. ESI-MS (m/z) for C54H86N8O17S (exact mass 1150.56): [M+H]+ calc. 1151.6, obs.
1151.7.
4.3.7. Boc-N-amido-(dPEG2)3-Phe-Arg(Pbf)-APdMG (10)
A solution containing 9 (23 mg, 0.02 mmol, 1 eq) and 1 (9 mg, 0.024 mmol, 1.2 eq) in DMF (1
mL) was prepared. To this DIEA (10 mg, 0.08 mmol, 14 μL, 4 eq) was added followed by
HATU (9 mg, 0.024 mmol, 1.2 eq). The reaction was monitored by HPLC, and completed within

30 minutes. The solvent was removed in vacuo, then resuspended in DCM. The DCM solution
was washed with water 4x, then evaporated leaving a tan oil. The crude material was purified by
reversed phase (C18) HPLC. The final product was isolated as a white solid (21 mg, 70% yield).
Analytical HPLC: Figure S6. ESI-MS (m/z) for C72H102ClFN12O18S (exact mass 1508.68):
[M+H]+ calc. 1509.7, obs. 1509.7; [M+2H]2+ calc. 755.4, obs. 755.0.
4.3.8. Boc-N-amido-(dPEG2)3-Phe-Arg(Pbf)-dPEG2APdMG (11)
A solution containing 9 (30 mg, 0.026 mmol, 1 eq) and 2 (18 mg, 0.034 mmol, 1.3 eq) in DMF
(1 mL) was prepared. To this DIEA (14 mg, 0.1 mmol, 18 μL, 4 eq) was added followed by
HATU (13 mg, 0.034 mmol, 1.3 eq). The reaction was monitored by HPLC, and completed
within 30 minutes. The solvent was removed in vacuo, then resuspended in DCM. The DCM
solution was washed with water 4x, then evaporated leaving a tan oil. The crude material was
purified by reversed phase (C18) HPLC. The final product was isolated as a white solid (28 mg,
65% yield). Analytical HPLC: Figure S7. ESI-MS (m/z) for C79H115ClFN13O21S (exact mass
1667.77): [M+2H]2+ calc. 834.9, obs. 834.7.
4.3.9. H2N-(dPEG2)3-Phe-Arg-APdMG (12)
TFA/water (9:1, 1 mL) was added 10 (21 mg, 0.014 mmol) and left at room temp for 1 hour. The
reaction was evaporated then dissolved in ACN/water, frozen and lyophilized leaving a white
solid. The material was used without further purification. The final product was left as a white
solid. Analytical HPLC: Figure S8. ESI-MS (m/z) for C54H78ClFN12O13 (exact mass 1156.55):
[M+H]+ calc. 1157.6, obs. 1157.8; [M+2H]2+ calc. 579.3, obs. 579.1.
4.3.10. H2N-(dPEG2)3-Phe-Arg-(dPEG2)-APdMG (13)
TFA/water (9:1, 1 mL) was added 11 (28 mg, 0.017 mmol) and left at room temp for 1 hour. The
reaction was evaporated then dissolved in ACN/water, frozen and lyophilized leaving a white

solid. Analytical HPLC: Figure S9. ESI-MS (m/z) for C61H91ClFN13O16 (exact mass 1315.64):
[M+H]+ calc. 1316.7, obs. 1316.5; [M+2H]2+ calc. 658.6, obs. 658.5.
4.3.11. S-Acetyl-mercaptoacetamido-(dPEG2)3-Phe-Arg-APdMG (14)
A solution containing 12 (5 mg, 0.004 mmol, 1 eq) and DIEA (1.5 mg, 0.012 mmol, 2 μL, 3 eq)
in DMF (200 μL) was prepared. SAMA-OPfp (2 mg, 0.006 mmol, 1.5 eq) in DMF (100 μL) was
then added to the solution, and allowed to react for 1 hour. The solvent was removed in vacuo
then purified by reversed phase HPLC. 3 mg of a white solid was recovered (60% yield).
Analytical HPLC: Figure S10. ESI-MS (m/z) for C58H82ClFN12O15 (exact mass 1272.54):
[M+H]+ calc. 1273.6, obs. 1273.3; [M+2H]2+ calc. 637.3, obs. 637.6.
4.3.12. S-Acetyl-mercaptoacetamido-(dPEG2)3-Phe-Arg-dPEG2APdMG (15)
A solution containing 13 (5 mg, 0.004 mmol, 1 eq) and DIEA (1.5 mg, 0.012 mmol, 2 μL, 3 eq)
in DMF (200 μL) was prepared. SAMA-OPfp (2 mg, 0.006 mmol, 1.5 eq) in DMF (100 μL) was
then added to the solution, and allowed to react for 1 hour. The solvent was removed in vacuo
then purified by reversed phase HPLC. 3 mg of a white solid was recovered (53% yield).
Analytical HPLC: Figure S11. ESI-MS (m/z) for C65H95ClFN13O18S (exact mass 1431.63):
[M+2H]2+ calc. 716.8, obs. 716.7.
4.3.13. Mercaptoacetamido-(dPEG2)3-Phe-Arg-APdMG, Phe-Arg-APdMG (3)
2 mg of 14 was dissolved in 100 μL of water/MeOH (1:1), to which 2 uL of 1 N NaOH was
added. After 15 minutes, 2 uL of 1 M HCl was added to neutralize. The solution was used
directly for subsequent experiments or conjugations. Analytical HPLC: Figure S12. ESI-MS
(m/z) for C56H80ClFN12O14S (exact mass 1230.53): [M+H]+ calc. 1231.5, obs. 1231.4; [M+2H]2+
calc. 616.3, obs. 616.3.

4.3.14. Mercaptoacetamido-(dPEG2)3-Phe-Arg-dPEG2APdMG, Phe-Arg-dPEG2APdMG
(4)
2 mg of 15 was dissolved in 100 μL of water/MeOH (1:1), to which 2 uL of 1 N NaOH was
added. After 15 minutes, 2 uL of 1 M HCl was added to neutralize. The solution was used
directly for subsequent experiments or conjugations. Analytical HPLC: Figure S13. ESI-MS
(m/z) for C63H93ClFN13O17S (exact mass 1389.62): [M+H]+ calc. 1390.6, obs. 1390.5; [M+2H]2+
calc. 695.8, obs. 695.7.
4.3.15. Fmoc-Lys(Mtt)-PABOH (16)
A solution of Fmoc-Lys(Mtt)-OH (748 mg, 1.2 mmol, 1 eq) and para-amino benzyl alcohol (300
mg, 2.4 mmol, 2 eq) in DMF (5 mL) was prepared. DIEA (465 mg, 3.6 mmol, 630 μL, 3 eq) was
added followed by a solution of HATU (502 mg, 1.3 mmol, 1.1 eq) in DMF (2 mL). The reaction
was complete within 30 minutes and as determined by HPLC/LCMS. The solvent was partially
removed in vacuo, and extracted with ethyl acetate/water. The ethyl acetate layer was washed 4x
with water, then evaporated leading to an orange solid. The crude material was purified on a 40 g
RediSep Rf normal phase cartridge using a linear gradient of hexane and ethyl acetate. The final
1
product was isolated as a white solid (850 mg, 97% yield). H-NMR (500 MHz, CDCl3): δ 7.96
(s, 1H), 7.73 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.5 Hz, 2H), 7.47 – 7.41 (m, 6H), 7.39 – 7.20 (m,
12H), 7.18 – 7.11 (m, 2H), 7.05 (d, J = 7.9 Hz, 2H), 5.29 (s, 1H), 4.63 (s, 1H), 4.44 (d, J = 6.4
Hz, 2H), 2.28 (s, 3H), 2.11 (t, J = 6.9 Hz, 2H), 1.88 (s, 1H), 1.58 (s, 5H), 1.51 (s, 1H), 1.38 (s,
2H). 13C-NMR (500 MHz, CDCl3): δ 146.36, 143.22, 141.32, 135.70, 128.57, 128.52, 128.49,
127.79, 127.74, 127.12, 126.14, 124.92, 120.16, 120.02, 77.27, 77.02, 76.76, 70.62, 64.92, 60.41,
47.16, 43.30, 30.57, 23.43, 21.07, 20.93, 14.21, 0.01. ESI-MS (m/z) for C48H47N3O4 (exact mass
729.36): [M+H]+ calc. 730.4, obs. 730.2.

4.3.16. Fmoc-Phe-Lys(Mtt)-PABOH (17)
The Fmoc group was removed from 16 (425 mg, 0.6 mmol, 1 eq) using 9 mL of 30% piperidine
in DMF for 10 minutes. The solvent was removed in vacuo, and the resulting oil was
resuspended in 10 mL DMF A solution of Fmoc-Lys(Mtt)-OH (748 mg, 1.2 mmol, 1 eq) and
para-amino benzyl alcohol (300 mg, 2.4 mmol, 2 eq) in DMF (5 mL) was prepared. DIEA (465
mg, 3.6 mmol, 630 μL, 3 eq) was added followed by a solution of HATU (502 mg, 1.3 mmol,
1.1 eq) in DMF (2 mL). The reaction was complete within 30 minutes and as determined by
HPLC/LCMS. The solvent was partially removed in vacuo, and extracted with ethyl
acetate/water. The ethyl acetate layer was washed 4x with water, then evaporated leading to an
orange solid. The crude material was purified on a 40 g RediSep Rf normal phase cartridge using
a linear gradient of hexane and ethyl acetate. The final product was isolated as a white solid (850
1
mg, 97% yield). H-NMR (500 MHz, CDCl3) δ 8.20 (s, 1H), 7.73 (d, J = 7.6 Hz, 2H), 7.50 (s,
2H), 7.48 – 7.34 (m, 8H), 7.34 – 7.20 (m, 11H), 7.20 – 7.07 (m, 7H), 7.04 (d, J = 8.1 Hz, 2H),
6.28 (s, 1H), 5.22 (s, 1H), 4.62 (s, 2H), 4.43 (dd, J = 10.7, 6.7 Hz, 1H), 4.32 (d, J = 11.5 Hz, 1H),
3.05 (s, 2H), 2.27 (s, 3H), 2.11 – 2.02 (m, 2H), 1.89 (s, 1H), 1.46 (d, J = 6.3 Hz, 1H), 1.26 (s,
2H). 13C-NMR (500 MHz, CDCl3) δ 146.35, 141.31, 136.97, 135.70, 129.10, 128.93, 128.57,
128.50, 127.82, 127.74, 127.71, 127.39, 127.12, 126.14, 124.91, 124.84, 120.10, 120.04, 77.28,
77.23, 77.02, 76.77, 70.60, 67.15, 64.96, 54.06, 47.08, 43.35, 31.30, 30.60, 23.52, 20.93. ESI-
MS (m/z) for C57H56N4O5 (exact mass 876.43): [M+H]+ calc. 877.4, obs. 877.3.
4.3.17. Fmoc-Phe-Lys(Mtt)-PABC-APdMG (19)
Compound 17 (420 mg, 0.5 mmol, 1 eq) was dissolved in anhydrous DCM (20 mL). Pyridine
(216 mg, 2.7 mmol, 5.4 eq) was added, followed by a solution of 4-nitrophenyl chloroformate
(180 mg, 0.9 mmol, 1.8 eq) in anhydrous DCM. The reaction proceeded for 2 hours at room