Tuning Isonitrile/Tetrazine Chemistry for Accelerated Deprotection and Formation of Stable Conjugates

Article abstract of DOI:10.1021/acs.joc.9b02522

The isocyano group is a valuable functionality for bioorthogonal reactions because it rapidly reacts with tetrazines to either form stable conjugates or release payloads from 3-isocyanopropyl groups. Here we provide mechanistic insights into the dissociative steps that follow the initial cycloaddition and analyze how structural modifications affect these processes. Three main outcomes of this study have important implications for designing such groups for bioorthogonal applications. First, anion-stabilizing substituents at C-2 of the 3-isocyanopropyl group promote β-elimination and accelerate deprotection. Second, tetrazines with bulky substituents form stable imine conjugates even with primary isonitriles that are otherwise rapidly hydrolyzed. Third, the elimination step is independent from hydrolysis to the aldehyde and instead can occur directly from the imine intermediate. These findings will allow tuning the structures of tetrazine and isonitrile reactants for application in bioorthogonal ligation and release chemistry.

Full text of DOI:10.1021/acs.joc.9b02522

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Article
Tuning Isonitrile/Tetrazine Chemistry for Accelerated
Deprotection and Formation of Stable Conjugates
Minghao Xu, Titas Deb, Julian Tu, and Raphael M. Franzini
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 04 Nov 2019
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Tuning Isonitrile/Tetrazine Chemistry for Accelerated Deprotection
and Formation of Stable Conjugates
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Minghao Xu,[a,b],† Titas Deb,[a] ,† Julian Tu,[a] Raphael M. Franzini[a],*
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[a] Department of Medicinal Chemistry, University of Utah, 30 S 2000 E, Salt Lake City, UT
84112, USA
[b] Current address: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901
Atlantic Drive, Atlanta, GA 30332, USA.
† These authors contributed equally
* To whom correspondence should be addressed: Raphael.franzini@utah.edu
Abstract
The isocyano group is a valuable functionality for bioorthogonal reactions because it rapidly reacts
with tetrazines to either form stable conjugates or release payloads from 3-isocyanopropyl groups.
Here we provide mechanistic insights into the dissociative steps that follow the initial
cycloaddition and analyze how structural modifications affect these processes. Three main
outcomes of this study have important implications for designing such groups for bioorthogonal
applications. First, anion-stabilizing substituents at C-2 of the 3-isocyanopropyl group promote β-
elimination and accelerate deprotection. Second, tetrazines with bulky substituents form stable
imine conjugates even with primary isonitriles that are otherwise rapidly hydrolyzed. Third, the
elimination step is independent from hydrolysis to the aldehyde and instead can occur directly
from the imine intermediate. These findings will allow tuning the structures of tetrazine and
isonitrile reactants for application in bioorthogonal ligation and release chemistry.
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Introduction
Several release reactions that work under physiological conditions have been developed1-14
establishing a dissociative bioorthogonal chemistry toolbox.15, 16 We are especially intrigued by
the isocyano group that, despite its exceptional structural compactness and unique reactivity,17, 18
remains remarkably underused as a bioorthogonal functionality. Isocyanides react rapidly with
tetrazines (rates of up to 4.0ꢀm-1ꢀs-1 in PBS/DMSO (90:10) at 37ꢀ°C) in a (4+1) cycloaddition to
form 4-aminopyrazoles (Fig. 1).19, 20 This reaction is applicable to both labeling and release uses.
Leeper et al. showed that tuning the structure of the isocyano reactant allows forming stable
conjugates with tetrazines.21-23 Our group recently developed biocompatible protecting groups
based on this reaction. 3-isocyanopropyl (ICPr) and 3-isocyanopropyl-1-carbamoyl (ICPrc) groups
react with tetrazines to spontaneously liberate phenols and amines under physiological conditions.3
Our unexpected discovery that steric attractions in the transition state of the (4+1) cycloaddition
accelerate the transformation further expanded the usefulness of this bioorthogonal reaction.24
However, mechanistic questions remain and optimization of reactants will be needed to access the
full potential of the chemistry.
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Figure 1: Mechanism of tetrazine-mediated removal of 3-isocyanopropyl groups. Red lines: steps analyzed in this
study; dotted line: hypothetical transformation.
Here we analyze the dissociative steps of the transformations following the cycloaddition (Fig. 1).
The tetrazine-triggered release of leaving groups from ICPr structures occurs in a cascade of steps.
Release of N2 and the formation of a five-membered ring with an exocyclic C=N bond (not shown
in figure) follows the initial (4+1)-cycloaddition.19 For most isonitriles the intermediate
tautomerizes to an imine of 4-aminopyrazole (imine intermediate in Fig. 1). We previously
hypothesized that hydrolysis of this imine intermediate into an aldehyde and 4-aminopyrazole
elicits the release of leaving groups by a spontaneous β-elimination reaction.25, 26 However, the
later steps in this postulated mechanism await rigorous examination. Furthermore, structure-
reactivity analysis might allow improving critical aspects of the reaction. First, serum albumin is
required for rapid payload release from ICPr groups.3 Chemical changes favoring elimination
would be helpful to ensure rapid and condition-independent release. Second, identifying tetrazines
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that form stable adducts with simple primary isonitriles would allow exploiting the minimal size
of the isocyano group in bioconjugation chemistry. Previously, the formation of stable adducts
required tertiary isonitriles or the presence of nearby carbonyls.23 Third, achieving release
independent of imine hydrolysis would avoid the formation of reactive acrolein side-products.
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The presented studies revealed that simple modifications of the 3-isocyanopropyl structure
accelerated release without decreasing the reactivity to tetrazines. Additionally, we discovered that
varying tetrazine substituents made it possible to tune the hydrolysis rate of the imine intermediate
and stabilize the conjugate. Finally, a mechanistic analysis unraveled that the release step could
occur independent of imine hydrolysis. The combined findings will guide the development of
isonitrile and tetrazine reagents for diverse uses in chemistry and in the life sciences.
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Results and Discussion
Accelerating β-elimination from tetrazine-activated 3-isocyanopropyl groups
Figure 2. Structures of probes to evaluate the effect of chemical modifications on the β-elimination step from
tetrazine-activated 3-isocyanopropyl groups. a) General scheme of tetrazine-mediated deprotection reaction. b)
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Structure and synthesis of fluorogenic naphthalimide used to study the release of amines and phenols. Reaction
conditions: i: NaCN, MeOH, 62%; ii: BMS (borane-dimethyl sulfide), dry THF, 20%;27 iii: TBDMSCl, NaH, dry
THF, 59%; iv: Phthalimide, PPh3, DEAD, dry THF, 50%; v: Hydrazine, EtOH, 30%;28 vi: ethyl formate, RT; vii:
Burgess reagent (yields for steps vi and vii: 55% for ICPPr-OH and 35% for ICPPr*-OH); viii: TsCl, TEA, dry THF,
59%; ix: N-(methyl-PEG4)-4-amino-1,8-naphthalimide,triphosgene, DIEA, dry toluene; x: N-(methyl-PEG4)-4-
hydroxy-1,8-naphthalimide, K2CO3, acetone, 24%.
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3-isocyanopropyl groups react with tetrazines to release amines (from carbamates) and phenols
under physiological conditions.3 Serum albumin catalyzes the β-elimination, and the release in
serum occurs in minutes.26 The rate-accelerating effect of albumin on β-elimination from
oxopropyl groups, presumably caused by a general-base effect from the lysine residues in
subdomain IIA of serum albumin, is well established. 25, 26, 29 However, the liberation step requires
hours to go to completion in PBS buffer.3 For certain applications, rapid release in the absence of
albumin will be critical.
We tested whether modifications at the C-1 and C-2 positions of the 3-isocyanopropyl scaffold
can facilitate the release of amines and phenols (Fig. 2). Of particular interest were substituents
predicted to promote β-elimination by decreasing the proton affinity at C-2.30 We designed 3-
isocyano-2-phenylprop-1-yl (ICPPr) moieties as rapid-release protecting groups responsive to
tetrazines (Fig. 2b). The design was inspired by the well-established pKa decrease of carbonyl α-
protons upon addition of phenyl substituents at the site of deprotonation (for example:
pKa(phenylacetone) = 19.8; pKa(acetone) = 26.5).31 The tetrazine-induced elimination from 3-
isocyano-1-phenylprop-1-yl (ICPPr*) was assessed for comparison. The β-elimination from 3-
ketoalkyl groups has been previously studied25 and was not further investigated. As reporter
probes, we prepared ICPr-modified naphthalimide dyes (Fig. 2b) because they undergo a
characteristic bathochromic shift upon deprotection of amine/hydroxy groups on C-4.32
Reagents to access ICPPr-protected molecules were synthesized from 3-amino-2-phenylpropan-1-
ol (Fig. 2b), which was prepared in a three-step literature procedure from 2-phenylpropane-1,3-
diol.28 Formylation of 3-amino-2-phenylpropan-1-ol (ethyl formate, room temperature) and
dehydration with Burgess reagent,3 afforded 3-isocyano-2-phenylpropan-1-ol (ICPPr-OH; 55%
yield, two steps). Reacting ICPPr-OH with in situ generated 4-isocyanato-naphthalimide3 provided
reporter probe ICPPrc-NH-NA (Fig. 2b; ICPPrc: 3-isocyano-2-phenylpropylcarbamoyl) for
testing the tetrazine-mediated deprotection of amines. The naphthalimide dye contained a PEG4-
modification on the imide to enhance water solubility. The corresponding 3-isocyano-1-
phenylpropylcarbamoyl (ICPPrc*) derivative of 4-amino naphthalimide (Fig. 2b) was synthesized
analogously by reacting the dye-isocyanate with 3-isocyano-1-phenylpropan-1-ol, which was
generated by formylation and dehydration (35% yield, two steps) of 3-amino-1-phenylpropan-1-
ol27 (Fig. 2b). To synthesize ICPPr-modified phenols, ICPPr-OH was converted into the tosyl
compound ICPPr-Tos (TosCl, triethylamine, 59 % yield). Alkylation of 4-hydroxy naphthalimide
with ICPPr-Tos (K2CO3, acetone) yielded ICPPr-O-NA in 24 % yield. All conjugates were stable
for >72 h in diluted human serum (PBS:serum, 1:1 (v/v), T = 37 °C; Fig. S1 in the SI).
The elimination of amines and phenols from ICPPrc-NH-NA and ICPPr-O-NA following the
reaction with PEG4-3,6-di-2-pyridyl-1,2,4,5-tetrazine (PEG-DPTz)3 was monitored
spectrophotometrically by measuring the emergence of the characteristic absorbance band of 4-
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amino/4-hydroxy naphthalimide (λabs(ICPPrc-NH-NA) = 435 nm, c = 0.1 mM; λabs(ICPPrc-O-NA)
= 445 nm, c = 0.2 mM) and PEG-DPTz (c = 1 mM or 2 mM) in PBS:DMSO (4:1, v/v; Fig. 3).
Elimination rates were calculated using a sequential first-order rate law (Equation 1 in the
Supporting Information) using extrapolated bimolecular rate constants (Fig. 3d). As designed, the
phenyl-group at the C-2 position significantly accelerated the release step (Fig. 3b). The rate
constant of dye release from ICPPrc-NH-NA was 13.4-fold higher (kelim = (9.56 ± 0.56) × 10-4 s-1,
t1/2 = 0.20 h) than that for the ICPr-derivative ICPrc-NH-NA (kelim = (7.13 ± 0.18) × 10-5 s-1, t1/2 =
2.70 h). A significant acceleration (9.1-fold) of the elimination step by the C-2 phenyl group was
also observed for the phenolic dye (kelim(ICPPr-O-NA) = (1.47 ± 0.01) × 10-3 s-1, t1/2 = 0.13 h;
kelim(ICPr-O-NA) = (1.61 ± 0.08) × 10-4 s-1, t1/2 = 1.20 h). Release yields were near-quantitative
for both derivatives (98 ± 3 % for ICPPrc-NH-NA and 96 ± 3 % for ICPPr-O-NA; ICPPrc-NH-
NA and ICPPr-O-NA (c = 1 mM) and DPTz (c = 3 mM), in PBS: DMSO (1:3, v/v), T = 37 °C, t
= 3 h; Fig. 3c). Under these conditions, the release step occurs on the same timescale as the
cycloaddition step, and it therefore is conceivable that the cycloaddition is the rate-limiting step
and that the actual release step is faster. Control experiments with increased tetrazine
concentrations to accelerate the bimolecular step provided comparable values for kelim (not shown)
establishing that the measured rates correctly reflect the kinetics of the elimination step.
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Figure 3. Effect of phenyl groups at C-2 of 3-isocyanopropyl groups on the tetrazine-induced release of naphthalimide
dyes. a) Reaction schemes. b) Kinetics of the emergence of free naphthalimide dyes determined
photospectrometrically. The phenyl groups accelerated the release of 4-amino/4-hydroxy napthalimides from ICPPr-
derivatives relative to ICPr-derivatives. c) Quantification of amine and phenol release yields from ICPPr-modified
reporter probes reacting with PEG-DPTz. d) Dependence of the cycloaddition rate of 3-isocyanopropyl derivatives
with PEG-DPTz on the water content. Phenyl groups at the C-2 position had no statistically significant effect on the
bimolecular reaction step. (Experimental conditions: PBS [pH 7.4]:DMSO 4:1 (unless stated otherwise, v/v, T =
37°C).
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The accelerated elimination was achieved without affecting the rate of the bimolecular reaction of
the isocyano group with tetrazines. The measured bimolecular rate constants were quasi-identical
for ICPPr- and ICPr-derivatives with carbamate and phenol leaving groups at several solvent
compositions (Fig. 3d; Table S1 in the Supporting Information). This result established that it is
possible to modulate the rates of the cycloaddition and the elimination steps independently.
Interestingly, the reaction rates exhibited a quasi-linear dependence on the water content in the
tested range (from 20% to 90% content PBS, v/v; T = 37 ˚C, Fig. 3d).
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The effect of introducing a phenyl group at C-1 on the elimination and the bimolecular steps was
modest (Fig. 4). The rate of elimination measured for ICPPrc*-NH-NA with PEG-DPTz of kelim
=
(4.93 ± 0.03) × 10-5 s-1 (t1/2 = 3.91 h), was 31 % slower than for the unmodified carbamate dye
ICPrc-NH-NA (Fig. 4b). This result aligned with our expectations because the effect of the C-1
substituent on the acidity of the carbonyl α-proton is expected to be modest. The release yield of
the naphthalimide from ICPPrc*-NH-NA is near-quantitative (97 ± 5 %; Fig. 4c) and the
bimolecular rate (at 30% PBS in DMSO) was near-identical to that of ICPrc-NH-NA (Table S1 in
the Supporting Information). Previous experiments revealed that serum albumin catalyzes the
elimination step from ICPr-groups.3 The C-1 phenyl group did not obstruct the release-accelerating
effect of serum on carbamate elimination and in diluted serum the dye release occurred in minutes
(Fig. 4b; ICPPrc*-NH-NA, ICPrc-NH-NA (c = 0.1 mM) and PEG-DPTz (c = 1 mM) in
PBS:DMSO (4:1, v/v) and Serum : PBS (1:1, v/v)). The outcome of these experiments has
significant implications for designing probes and drug-release reagents. It establishes the C-1
position as a possible branching point for introducing handles for bioconjugation without
interfering with the cycloaddition and release steps.
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Figure 4. Effect of phenyl groups at C-1 of 3-isocyanopropyl groups on the tetrazine-induced release of carbamate
groups. a) Reaction schemes. b) Kinetics of the emergence of free naphthalimide dye determined
photospectrometrically. The phenyl group only modestly slowed the elimination step without interfering with the
catalytic effect of human serum albumin. c) Quantification of amine release from ICPPrc*-NH-NA reacting with
DPTz.
To further expand the scope of leaving groups, we tested the release of an aromatic thiol. An
ICPPr-derivative of 2-naphthalenethiol was synthesized by nucleophilic substitution on ICPPr-Tos
to afford ICPPr-S-NA. The release percentage was determined indirectly with Ellman’s reagent
and measured photospectrometrically. DPTz induced the release of the free thiol (65 ± 1 %; Fig.
S2 in the Supporting Information). Although the observed release yields were high, they were not
near-quantitative as observed for phenols and amines (Fig. 3). Possibly, part of the released 2-
nitro-5-thiobenzoic acid is alkylated by a downstream product (2-phenylacrolein or derivatives) of
the ICPPr-release reaction, leading us to underestimate the actual release yield. Nonetheless, the
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presented data clearly indicates that ICPPr-moieties can release diverse functional groups upon
reaction with tetrazines.
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In summary, the data that phenyl (as a carbanion-stabilizing substituent at the C2 position)
accelerates payload release from ICPr-derivatives without negatively impacting the cycloaddition
rates and release yields. This observation established the groundwork for developing tetrazine-
responsive protecting groups with accelerated elimination rate.
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Hydrolytic Stability of the Imine Intermediate
Figure 5. Hydrolysis of imine adduct formed by the reaction of isocyanides and tetrazines. a) Reaction sequence used
to evaluate the stability of the adduct in aqueous phase. b) Time dependence of conversion of imine intermediate into
butyraldehyde and 4-aminopyrazole; 1H NMR spectra are shown in Figs. S6, S7, S13 in the Supporting Information.
(Conditions: DMSO-d6:D2O (1:1 v/v), T = 37 °C)
The tetrazine-mediated conversion of isonitriles to carbonyls involves the hydrolysis of an imine
intermediate (Fig. 1).19, 23 For bioorthogonal chemistry applications, it is important to control the
rate of imine hydrolysis. For labeling applications, the adduct needs to be stable. Leeper et al. used
tertiary isonitriles and methyl 3-isocyanopropionate to overcome the lability of the isonitrile-
tetrazine adduct.23 Slow imine hydrolysis on the other hand could impede ICPr-deprotection
reactions. We therefore investigated the dependence of imine hydrolysis on the tetrazine
substituents.
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For these studies, we generated adducts of n-butyl isocyanide (n-BuNC) with tetrazines in DMSO-
d6 and monitored hydrolysis by 1H NMR following dilution with D2O (Fig. 5a). First, we assessed
the effect of substituents with different electronic properties on the persistence of the imine
intermediate. In agreement with results by Leeper et al.23 even the traces of water present in the
DMSO-d6 solvent used for the NMR studies were sufficient to partially hydrolyze the imine formed
with DPTz to the aldehyde and 4-aminopyrazole (Fig. S3 in the Supporting Information), and a
similar result was obtained for 3,6-di-2-pyrimidyl-1,2,4,5-tetrazine (Fig. S4 in the Supporting
Information). Therefore, adducts of simple alkyl isocyanides and these tetrazines hydrolyze
rapidly. The corresponding imine adduct of n-BuNC and 3,6-diphenyl-1,2,4,5-tetrazine persisted
in DMSO-d6 and hydrolyzed upon addition of D2O over a period of 2 h (Fig. S5 in the Supporting
Information). These results show that electron-withdrawing tetrazine substituents favor rapid
hydrolysis of the imine adduct.
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To assess steric effects of tetrazine substituents, we monitored imine hydrolysis for four symmetric
dialkyl-tetrazines with methyl (Me2Tz), ethyl (Et2Tz), isopropyl (iPr2Tz), and tert-butyl (tBu2Tz)
groups (Fig. 5b). The imine adducts of n-BuNC and Me2Tz and Et2Tz gradually hydrolyzed and
reached an equilibrium within an hour under the given conditions (DMSO-d6:D2O (1:1 v/v), T =
37 °C, Figs. S6, S7 in the Supporting Information). The rate of the dissociation of the adduct is
(8.77 ± 0.99) × 10-4 s-1 for Me2Tz and (4.73 ± 0.90) × 10-4 s-1 for Et2Tz (Fig. S8, Table S3 in the
Supporting Information). The formation of an equilibrium was confirmed by temperature cycling
experiments in which the ratio of imine to aldehyde products reproducibly adopted temperature-
dependent values (Me2Tz, T = 20 °C: Kd = (3.56 ± 0.25) × 10-3 M; T = 37 °C: Kd = (5.62 ± 0.99)
× 10-3 M; T = 50 °C: Kd = (1.61 ± 0.24) × 10-2 M; Figs. S9-S10 in the Supporting Information).
At the micromolar concentrations typical for chemical biology experiments, the dissociation would
be expected to go to completion given these equilibrium constants. Reaction of n-BuNC with
iPr2Tz resulted in two imine intermediates that showed distinctly different hydrolysis rates (Figs.
S11, S12 in the Supporting Information). The adduct of tBu2Tz and nBuNC is remarkably stable
(Fig. 5b). After 2 h in the presence of 50 % D2O, the imine signal was unchanged. Gradual imine
hydrolysis occurred reaching 18 ± 3 % after 72 h (Fig. S13 in the Supporting Information). The
stability of the adduct of a primary isonitrile with tBu2Tz was corroborated in 80% PBS:DMSO
by HPLC analysis (Fig. S14 in the Supporting Information) using a water-soluble isonitrile
(CH3O-PEG2-NC, structure shown in the Supporting Information).
In summary, the data demonstrated that tetrazine substituents modulate the hydrolysis rate of imine
intermediates. Adducts of isonitriles and tBu2Tz are stable for days, which allows obviating the
need of structurally engineering the isonitriles for bioconjugation. This possibility is especially
appealing because we have shown that bis-tert-butyl tetrazines allow for triple-orthogonal protein
labeling schemes.24
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Mechanistic analysis of the elimination step
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Figure 6. Mechanism of ICPr-removal by tetrazines. a) Possible reaction pathways. Hydrolysis of the imine
intermediate may generate a propanal that spontaneously eliminates the leaving group. Alternatively, release may
occur directly from the imine (or iminium) intermediate. b) Structures of probe molecules. c) Fluorescence monitoring
of resorufin release from ICPr-res precursor after reaction with DPTz (red trace) and tBu2Tz (blue trace) (ICPr-res (5
µM), tetrazine (100 µM) in PBS pH 7.4 : DMSO (4:1) at 37 ˚C). c) Time-dependent H NMR analysis of tBu2Tz-
induced release of phenoxyacetic acid from ICPr-protected precursor (ICPr-POA (5 mM), tBu2Tz (10 mM) in DMSO-
1
d6: D2O (4:1) at room temperature).
The finding that tetrazine substituents influence the hydrolysis of the imine intermediate raises
questions concerning the β-elimination step. The design of ICPr-chemistry assumed rapid
hydrolysis of the imine intermediate to form a 3-propanal species followed by spontaneous
β-elimination (Fig. 6a).3 According to this mechanism, tetrazines forming long-lived imines would
release payloads slowly. It is alternatively possible that release occurs directly from the imine (or
iminium) intermediate, and β-elimination from imines has been postulated.33
To test this possibility, we examined the liberation of a resorufin fluorophore from an ICPr-caged
precursor3 (ICPr-res, Fig. 6b). ICPr-res (5 µM) and tetrazine (100 µM) were incubated in PBS pH
7.4 : DMSO (4:1 v/v) at 37 ˚C and emergence of the resorufin fluorescence was measured as a
function of time (λex = 590 nm; λem = 610 nm; Fig. 6c). A rapid fluorescence increase was observed
with DPTz as expected. Importantly, emergence of a distinct resorufin signal was apparent for the
reaction with tBu2Tz even though the hydrolysis studies showed that the corresponding imine is
stable in the given timeframe under these conditions (Fig. 5). This outcome provides evidence that
resorufin release occurs from the imine/iminium intermediate. The slower uncaging of resorufin
with tBu2Tz relative to DPTz agrees with the different reaction rates of the cycloaddition step.24
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Only low quantities of resorufin were generated in control experiments without tetrazine (Fig. 6c)
confirming that resorufin release is tetrazine-mediated.
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NMR experiments were performed to verify the hydrolysis-independent removal of ICPr-groups
(Fig. 6d). We synthesized an ICPr-derivative of phenoxyacetic acid (ICPr-POA; Fig. 6b) having
a characteristic 1H NMR signal with a chemical shift at 4.74 ppm. ICPr-POA was synthesized in
three steps from 3-aminopropanol. Formylation of 3-aminopropanol (ethyl formate, 45 ˚C) was
followed by esterification with phenoxyacetic acid (EDC, DMAP, room temperature) and
dehydration to the isocyanide (tosyl chloride, room temperature), which yielded ICPr-POA (30%
yield, three steps). Time-dependent changes of the 1H NMR spectra were recorded for samples of
ICPr-POA (5 mM) and excess tBu2Tz (10 mM) in DMSO-d6:D2O (4:1) at 25 ˚C (Figs. S15-S16
in the Supporting Information). The signals linked to the ICPr-POA starting material progressively
disappeared, giving rise to an intermediate whose 1H NMR spectra agreed with that of the imine
structure predicted by the mechanism. The intermediate gradually disappeared and a peak at 4.61
ppm emerged that coincided with that of a phenoxyacetic acid standard in this NMR-solvent
system. Aldehyde signals were undetectable at any time of the measurement; instead distinct olefin
1H NMR signals associated with a side product, plausibly the α,β-unsaturated imine, emerged in
parallel with the release of phenoxyacetic acid, but disappeared as the reaction continued. The
experimental results did not allow to make qualified conclusions on the formed side product.
Broadening of the tert-butyl group at later time points suggested the formation of a complex
mixture of aminopyrazole derivatives of possibly macromolecular nature.
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The combined outcomes provide compelling evidence that elimination of leaving groups can occur
directly from the imine/iminium intermediate. ICPr-chemistry is therefore hydrolysis-independent
and may occur without generating acrolein side products.
Conclusions
This study provides important information on structure-activity relationships and mechanism of
the tetrazine-induced removal of ICPr-groups. Three key conclusions result from the series of
experiments. First, modifications that promote deprotonation at the C-2 position accelerate the
elimination of payloads. Second, tetrazines with bulky substituents (-tBu) form stable adducts with
primary isonitriles. Third, payload release can occur independently from imine hydrolysis. Each
of these outcomes has important implications for practical translations.
The accelerating effect of the C-2 phenyl group on the β-elimination step indicates that it is
possible to design ICPr-derivatives that, independent from albumin, release payloads rapidly. This
outcome is important for many applications where the reaction has to occur without the presence
of serum albumin. The ICPPr design establishes the basic principle for rate enhancement and
provides guidance for future design; one can easily envision other modifications of the ICPr-group
that would further accelerate tetrazine-mediated deprotection. Boosting the elimination step may
additionally open up the possibility of applying ICPr-chemistry to the bioorthogonal unmasking
of functional groups that are more difficult to eliminate than phenols and carbamates.
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The formation of stable adducts by the reaction between n-BuNC and tBu2Tz suggests that one
can use this chemistry to label a much broader range of isonitriles than previously thought possible.
This result is significant because it obviates the need for engineering the isonitrile moiety (e.g.
tertiary isonitriles23) and allows fully exploiting the isocyano functionality as the smallest
bioorthogonal reactive group. Such chemistry could find widespread use. For example,
carbohydrates with handles that are even smaller than those previously used for modifying sugars
that can be metabolically incorporated into glycans21, 22 could be used. Moreover, the structural
compactness of the isocyano group is ideal for incorporation into proteins by codon expansion
techniques and would minimally disrupt the structures of the biomacromolecules. The chemistry
could also be useful for the discovery of isocyano-containing natural products. Recently, isonitrile-
tetrazine chemistry was used in tandem with NaBH3CN reduction of the imine to isolate such
molecules.34 With sterically encumbered tetrazines, the reduction step could become unnecessary.
The possibility to precisely tune the half-life of isonitrile-tetrazine adducts may open new
possibilities in the biosciences.
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An important outcome was the discovery that elimination of the payloads can occur from the imine
intermediate. The fate of the imine of the 4-aminopyrazole following the reaction could not be
determined from the NMR results and are likely dependent on the exact reaction conditions.
Nevertheless, this result has significant implications. First, this result suggests that hydrolysis of
the imine is not rate-limiting for release. Second, it means that release can occur independent of
the structure of the tetrazine (i.e. even for tetrazines that form imine adducts that are inert to
hydrolysis). Third, the outcome is important for drug delivery applications. For example, Royzen
and Mejia Oneto et al. recently reported that implanted tetrazine-modified hydrogels combined
with reactive prodrugs enabled the site-specific delivery of doxorubicin35 and antibiotics.36
Delayed drug release relative to the dissociation from the hydrogel would lead to diffusion of the
prodrug away from the site of interest and diminish the spatial resolution of such an approach.
Drug release from the imine intermediate overcomes this problem. Importantly, it can also avoid
the formation of the acrolein side-product that was previously considered a primary disadvantage
of ICPr-chemistry. Although it is possible that the formed side product may be intrinsically toxic,
as it is retained on the hydrogel, or any other drug carrier, its detrimental effect will likely be
greatly diminished. The therapeutic prospect of this chemistry will be explored in future studies.
In summary, the presented data provides detailed insights on the dissociative steps of tetrazine-
induced ICPr-deprotection chemistry. The obtained knowledge will be valuable for designing
applications based on this reaction in chemical biology and drug delivery.
Experimental Section
Materials and Instrumentation
All chemical reagents and solvents were obtained from commercial sources (Sigma-Aldrich, Alfa-
Aesar, Combi-Blocks, Acros-Organic, TCI) and used without further purification. Thin-layer
chromatography (TLC) analysis was carried out to monitor the process of reactions. Purification
of compounds was performed by column chromatography with silica gel 300-400 mesh. 1H NMR
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and 13C NMR spectra were recorded on a Varian Mercury-400 or Varian Inova-500 spectrometer
as indicated with chemical shifts expressed as ppm (in CDCl3, MeOD-d4 or DMSO-d6) using
Me4Si (TMS) as internal standard.
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Exact Mass Spectra were measured by the University of California, Riverside Chemistry Mass
Spectrometry Facility. All analytical preparative HPLCs were performed on a Dionex
Ultimate3000 equipped with autosampler, diode array detector and robotic fraction collector
(Dionex Thermo Scientific, USA) using a Gemini C18 column (5 μM, 250×10 mm, Phenomenex,
USA) or Luna C18 column (5 μM, 150×2.0 mm, Phenomenex, USA). UV-VIS photospectrometic
kinetic measurements were performed on a microplate reader SpectramaX M5 (Molecular Device,
USA) in 96-well plates or 1 mL quartz cuvette.
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Synthetic Procedures
3-((tert-butyldimethylsilyl)oxy)-2-phenylpropan-1-ol. A solution of 2-phenylpropane-1,3-diol
(5 g, 33 mmol) in THF (25 mL) was added dropwise to a stirred and ice cooled suspension of NaH
(1.5 g, 36 mmol) in THF (15 mL). The ice bath was removed and reaction mixture was allowed to
warm to room temperature and kept at this temperature for 12 h. The reaction was quenched with
ice, diluted with EtOAc (300 mL) and washed with brine (2×150 mL). The separated organic layer
was dried with anhyd. Na2SO4, filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography (100% hexane) to afford the desired compound as a yellow
solid in a yield of 5 g (59%; Rf = 0.8 in hexane : EtOAc = 100:1, v/v). 1H NMR (400 MHz, CDCl3)
δ 7.33-7.30 (m, 2H), 7.24 (brs, 1H), 7.22-7.20 (m, 2H), 4.13-4.05 (m, 2H), 3.93-3.87 (m, 2H),
3.12-3.05 (m, 1H), 1.26 (t, J = 7.2 Hz, 2H), 0.90 (s, 9H), 0.06 (s, 6H). The 1H NMR data agreed
with the reported 1H NMR spectrum of this compound.28
2-(3-((tert-butyldimethylsilyl)oxy)-2-phenylpropyl)isoindoline-1,3-dione.
Triphenylphosphine (6.3 g, 24 mmol) and phthalimide (2.8 g, 19.2 mmol) were added sequentially
to a stirred and ice cooled solution of 3-((tert-butyldimethylsilyl)oxy)-2-phenylpropan-1-ol (4.4 g,
16 mmol) in THF (60 mL). To this mixture diethyl azodicarboxylate (3.34g, 19.2 mmol) in THF
(15 mL) was added dropwise. The ice bath was removed and reaction mixture was allowed to
warm to room temperature and kept at this temperature for 12 h. The reaction was quenched with
ice, diluted with Et2O (200 mL) and washed with sat. aq. NaHCO3 (2×150 mL) and brine (2×150
mL). The separated organic layer was dried with anhyd. Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatography (hexane : EtOAc = 10:1,
v/v) to afford the desired compound as a yellow oil in a yield of 3.2 g (50%; Rf = 0.6 in hexane :
EtOAc = 5:1, v/v). 1H NMR (400 MHz, CDCl3) δ 7.78-7.76 (m, 2H), 7.66-7.64 (m, 2H), 7.25-7.18
(m, 5H), 4.11-4.00 (m, 2H), 3.85-3.81 (m, 2H), 3.49-3.44 (m, 1H), 1.26 (t, J = 7.2 Hz, 2H), 0.81
1
1
(s, 9H), 0.09 (s, 6H). The H NMR data agreed with the reported H NMR spectrum of this
compound.28
3-Amino-2-phenylpropan-1-ol. Hydrazine hydrate (2 g, 40 mmol) was added to a stirred and ice
cooled solution of compound 2-(3-((tert-butyldimethylsilyl)oxy)-2-phenylpropyl)isoindoline-1,3-
dione (1.6 g, 4 mmol) in EtOH (40 mL) and the reaction mixture was reflux for 2 h. The reaction
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was quenched with ice, diluted with Et2O (200 mL) and washed with sat. aq. NaHCO3 (2×150
mL). The aqueous layer was extracted with Et2O (100 mL) and the combined organic layers were
washed with 1 M aq. HCl solution. The resulting acidic solution was then treated with 10 M aq.
NaOH until reaching pH 11 and washed with EtOAc (200 mL). The combined organic layers were
dried with anhyd. Na2SO4, filtered, and concentrated under reduced pressure to give the crude
1
compound as yellow oil in a yield of 185 mg (30%; Rf = 0.1 in EtOAc : MeOH = 5:1, v/v). H
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NMR (400 MHz, CDCl3) δ 7.36-7.30 (m, 3H), 7.24-7.22 (m, 2H), 4.10-4.04 (m, 2H), 3.99 (t, J =
7.6 Hz, 1H), 3.54-3.49 (m, 1H), 3.32-3.26 (m, 1H). HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for
C9H14NO 152.1070; Found 152.1095. The 1H NMR data agreed with the reported spectrum of this
compound.28
3-isocyanide-2-phenylpropan-1-ol (ICPPr-OH). Ethyl formate (850 mg, 12 mmol) was added
in portions to a stirred ice cooled solution of 3-amino-2-phenylpropan-1-ol (185 mg, 1.2 mmol).
The ice bath was removed and the reaction mixture heated to 50 oC for 2 h. Excess ethyl formate
was removed by rotary evaporation to afford the desired product as a colorless oil in a near-
quantitative yield (>95%). This compound decomposed upon storage and was immediately used
in the next step. Burgess reagent (350 mg, 1.5 mmol) was added in portions over 10 min to a
solution of the aforementioned intermediate in dry CH2Cl2 (2.5 mL). The solution was stirred at
room temperature under nitrogen atmosphere for 2 h. The mixture was diluted with CH2Cl2 (50
mL), washed with brine (50 mL), dried with anhyd. Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatography (Hexane : EA = 3:1, v/v)
to afford the desired compound as a yellow solid in a yield of 106 mg (55%; Rf = 0.3 in EtOAc :
hexane = 1:3, v/v). 1H NMR (400 MHz, CDCl3) δ 7.39-7.31 (m, 3H), 7.24-7.22 (m, 2H), 3.92-3.77
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(m, 2H), 3.73-3.68 (m, 2H), 3.12 (brs, 1H). C{1H} NMR (100 MHz, CDCl3) δ156.8, 156.7,
137.8, 128.9, 127.8, 127.7, 63.1, 46.9, 43.5, 43.5, 43.4. HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for
C10H12NO 162.0913; Found 162.0929.
3-isocyano-2-phenylpropan-1-tosylpropane (ICPPr-Tos). To a stirred solution of compound
ICPPr-OH (65 mg, 0.4 mmol) in dry THF (1 mL) was added dropwise over 30 minutes a solution
of tosyl chloride (114 mg, 0.6 mmol) and triethylamine (60 mg, 0.6 mmol) in dry THF (1 mL).
The solution was stirred at 0 oC under a nitrogen atmosphere for 4 hours. The reaction was diluted
with DCM (20 mL) and washed with brine (2×30 mL). The organic layer was dried over anhyd.
Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column
chromatography (DCM) to afford the desired compound as an oil in a yield of 75 mg (59%; Rf =
0.3 in DCM : hexane = 10:1, v/v). 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.0 Hz, 2H), 7.35-
7.32 (m, 5H), 7.16 (d, J = 8.0 Hz, 2H), 4.28-4.25 (m, 2H), 3.73 (t, J = 4.4 Hz, 2H), 3.29 (t, J = 6.4
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Hz, 1H), 2.45 (s, 3H). C{1H} NMR (100 MHz, CDCl3) δ 145.2, 135.6, 132.2, 130.0, 129.0,
128.4, 127.9, 127.6, 69.2, 43.9, 21.6, 14.1. HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C17H18NO3S
316.1002; Found 316.1005.
N-(methyl-PEG4)-4-(3-isocyanopropyl-2-phenylcarbamoyl)amino-1,8-naphthalimide
(ICPPrc-NH-NA). To a mixture of N-(methyl-PEG4)-4-amino-1,8-naphthalimide3 (48 mg, 0.12
mmol) and DIEA (44 mg, 0.36 mmol) in dry toluene (2.5 mL) was added a solution of triphosgene
(39 mg, 0.13 mmol) in dry toluene (1 mL) dropwise. The solution was heated to reflux for 3 h.
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After cooling to room temperature, the mixture was added a solution of ICPPr-OH (39 mg, 0.24
mmol) in dry CH2Cl2 (3 mL) and the reaction was stirred at room temperature for 12 h. The mixture
was concentrated under reduced pressure and the residue was purified by column chromatography
(DCM : MeOH = 20:1, v/v) to afford the desired compound as yellow oil in a yield of 21 mg (32%,
1
; Rf = 0.25 in DCM : MeOH = 20:1, v/v). H NMR (400 MHz, CD3OD) δ 8.54 (d, J = 7.6 Hz,
1H), 8.48 (t, J = 7.2 Hz, 2H), 8.14 (d, J = 7.6 Hz, 1H), 7.80-7.76 (m, 1H), 7.41-7.33 (m, 5H), 4.56-
4.53 (m, 2H), 4.37 (t, J = 6.0 Hz, 2H), 4.03-3.91 (m, 2H), 3.79 (t, J = 6.0 Hz, 2H), 3.66-3.64 (m,
2H), 3.58-3.56 (m, 2H), 3.51-3.46 (m, 5H), 3.43-3.41 (m, 4H), 3.29 (s, 3H). 13C{1H} NMR (100
MHz, CD3OD) δ 163.8, 163.3, 154.0, 140.9, 138.2, 132.0, 131.3, 129.73, 129.0, 128.8, 128.7,
128.5, 128.5, 128.0, 126.7, 124.3, 122.5, 118.7, 118.4, 117.5, 71.6, 70.2, 70.1, 70.0, 69.9, 67.3,
65.7, 58.4, 44.0. HRMS (ESI-TOF) m/z: [M+Na]+ Calcd. for C32H35N3O8Na 612.2316; Found
612.2365.
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N-(methyl-PEG4)-4-(3-isocyanopropyl-2-Phenol-1-oxy)-1,8-naphthalimide (ICPPr-O-NA).
To a mixture of N-(methyl-PEG4)-4-hydroxy-1,8-naphthalimide[2] (41 mg, 0.1 mmol) in acetone
(0.5 mL) was added anhydrous K2CO3 (22 mg, 0.16 mmol) and the mixture was allowed to stir at
room temperature for 15 minutes. ICPPr-Tos (60 mg, 0.2 mmol) dissolved in acetone (0.5 mL)
was added dropwise to the solution and the reaction mixture was allowed to stir at 50 ˚C for 12 h.
The solution was cooled to room temperature, solid residues were removed by filtration, and the
organic layer was concentrated under reduced pressure. Residues were purified by column
chromatography (DCM : MeOH = 30:1, v/v) to afford the desired compound as an orange oil in a
yield of 12 mg. (24%, ; Rf = 0.3 in DCM : MeOH = 20:1, v/v). 1H NMR (400 MHz, CDCl3) δ 8.61
(d, J = 7.2 Hz, 1H), 8.52 (d, J = 8.0 Hz, 1H), 8.47 (d, J = 8.4 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H),
7.45-7.37 (m, 5H), 7.06 (d, J = 8.0 Hz, 1H), 4.58 (d, J = 6.4 Hz, 2H), 4.43-4.40 (m, 2H), 4.04-4.00
(m, 2H), 3.80 (d, J = 6.4 Hz, 3H), 3.70-3.67 (m, 4H), 3.62-3.56 (m, 8H), 3.52-3.49 (m, 2H), 3.35
(s, 3H), 3.15-3.09 (m, 1H). 13C{1H} NMR (100 MHz, CDCl3) δ 164.3, 163.7, 159.1, 145.2, 136.7,
135.6, 133.2, 131.7, 130.0, 129.4, 129.2, 129.0, 128.4, 128.4, 128.2, 127.9, 127.6, 127.6, 126.2,
123.3, 122.5, 115.7, 106.0, 71.8, 70.5, 70.4, 70.0, 69.2, 69.0, 67.9, 44.4, 43.9, 21.6. HRMS (ESI-
TOF) m/z: [M+Na]+ Calcd. for C31H34N2O7Na 569.2258; Found 569.2295.
2-Cyano-1-phenylethanol. To a solution of styrene oxide (4 mL, 35 mmol) in MeOH (100 mL)
was added a solution of sodium cyanide (2 g, 100 mmol) in water (20 mL). The reaction mixture
was stirred for 12 h. The mixture was then quenched with water (80 mL) and a 2 aq. N HCl solution
(100 mL). CAUTION: evolution of hydrogen cyanide gas! The reaction mixture was diluted with
DCM (400 mL) and washed with brine (2×200 mL). The organic layer was dried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue was purified by column
chromatography (hexane : EA = 6:1, v/v) to afford the desired compound as an oil in a yield of 3
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g. (62%, Rf = 0.2 in hexane : EA = 6:1, v/v). H NMR (400 MHz, CDCl3) δ 7.41-7.36 (m, 5H),
5.05 (t, J = 6.4 Hz, 1H), 2.78 (d, J = 6.4 Hz, 2H). The 1H NMR data agreed with the reported 1H
NMR spectrum of this compound. 37
3-Amino-1-phenylpropan-1-ol. A solution of borane-dimethyl sulfide complex (2.2 mL, 23
mmol) in dry THF (10 mL) was added dropwise to a solution of 2-cyano-1-phenylethanol (3 g, 21
mmol) in dry THF (10 mL) at 0 ○C under a N2 atmosphere. The mixture was refluxed for 8 h. The
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reaction was quenched with ice water, diluted with EtOAc (200 mL) and washed with brine (200
mL). The combined organic layers were dried over anhyd. Na2SO4, filtered, concentrated under
reduced pressure. The residue was purified by column chromatography (EA : MeOH = 1:1, v/v)
to afford a colorless oil in a yield of 600 mg (20%, Rf = 0.15 in EA : MeOH = 1:1, v/v). 1H NMR
(400 MHz, CDCl3) δ 7.39-7.23 (m, 5H), 4.97 (d, J = 8.4 Hz, 1H), 2.78 (brs, 2H), 1.89-1.74 (m,
1
2H). HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C9H14NO 152.1070; Found 152.1062. The H
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NMR data agreed with the reported 1H NMR spectrum of this compound.27
3-isocyanide-1-phenylpropan-1-ol (ICPPr*-OH). ICPPr*-OH was prepared following the same
procedure as for ICPPr-OH. ICPPr*-OH was obtained as a colorless oil in a yield of 220 mg (35%,
Rf = 0.35 in EA : hexane = 1:3, v/v). 1H NMR (400 MHz, CDCl3) δ 7.38-7.31 (m, 5H), 4.90-4.86
13
(m, 1H), 3.67-3.59 (m, 1H), 3.47-3.39 (m, 1H), 2.08-2.03 (m, 1H). C{1H} NMR (100 MHz,
CDCl3) δ 156.0, 156.0, 155.9, 143.1, 128.7, 128.3, 128.1, 125.6, 70.6, 38.6, 38.5, 38.4, 37.9, 29.7.
HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C10H12NO 162.0913; Found 162.0905.
N-(methyl-PEG4)-4-(3-isocyanopropyl-3-phenylcarbamoyl)amino-1,8-naphthalimide
(ICPPrc*-NH-NA). ICPPrc*-NH-NA was prepared following the same procedure as for ICPPrc-
NH-NA. ICPPrc*-NH-NA was obtained as a yellow oil in a yield of 15 mg (35%, Rf = 0.35 in EA
: hexane = 1:3, v/v). 1H NMR (400 MHz, CDCl3) δ 8.52 (t, J = 8.0 Hz, 2H), 8.32-8.29 (m, 2H),
8.09 (brs, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.59-7.49 (m, 5H), 6.13-6.09 (m, 1H), 4.52-4.49 (m, 2H),
3.97 (t, J = 6.0 Hz, 2H), 3.87-3.85 (m, 2H), 3.78-3.68 (m, 6H), 3.63-3.59 (m, 5H), 3.55-3.53 (m,
13
2H), 3.42 (s, 3H), 2.63-2.37 (m, 2H). C{1H} NMR (100 MHz, CDCl3) δ163.9, 163.4, 157.0,
152.2, 138.8, 138.3, 132.1, 131.0, 128.8, 128.8, 128.3, 126.3, 126.2, 122.7, 117.3, 116.8, 74.7,
71.6, 70.5, 70.3, 70.3, 70.2, 70.0, 67.9, 58.8, 38.9, 38.2, 35.5, 29.5. HRMS (ESI-TOF) m/z:
[M+Na]+ Calcd. for C32H35N3O8Na 612.2316; Found 612.2371.
(3-isocyano-2-phenylpropyl)(naphthalen-2-yl)sulfane. To a mixture of 2-naphthalenethiol (6
mg, 0.033 mmol) in DMF (0.1 mL) was added anhydrous K2CO3 (17 mg, 0.12 mmol) and the
mixture was stirred at room temperature for 15 minutes. ICPPr-Tos (10 mg, 0.03 mmol) dissolved
in DMF (0.1 mL) was added dropwise and the reaction mixture was stirred at room temperature
for 12 h. Solid residues were removed by filtration, and the organic layer was concentrated under
reduced pressure and the residue purified by column chromatography (Hexane : EA = 10:1, v/v)
to afford the desired compound as an orange oil in a yield of 8 mg. (20%, ; Rf = 0.3 in Hexane :
EA = 10:1, v/v). 1H NMR (400 MHz, CDCl3) δ 7.82-7.76 (m, 4H), 7.50-7.31 (m, 6H), 7.22 (d, J
= 7.2 Hz, 2H), 3.87-3.76 (m, 2H), 3.43-3.40 (m, 2H), 3.17 (brs, 1H). 13C{1H} NMR (100 MHz,
CDCl3) δ 139.1, 133.6, 132.2, 132.0, 128.9, 128.8, 128.0, 127.7, 127.5, 127.5, 127.1, 126.7, 126.0,
45.8, 44.0, 36.2, 31.9, 29.7, 22.7. HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C20H18NS 304.1154;
Found 304.1154.
3-formamidopropyl 2-phenoxyacetate. Ethyl formate (311 mg, 4.2 mmol) was added in portions
to 3-aminopropanol (247.5 mg, 3.3 mmol) while stirring at 0 ℃. The solution was removed from
o
the ice-bath and was heated at 50 C for 2 h. Excess ethyl formate was removed by rotary
evaporation to afford the desired product as a colorless oil in near-quantitative yield (>95%). This
compound decomposed upon storage and was immediately used in the next step. To a solution of
phenoxyacetic acid (456 mg, 3 mmol) in dry DCM (5 mL), a solution of the aforementioned
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intermediate and DMAP (508 mg, 4.2 mmol) in dry DCM (5 mL) was added at 0 ℃ under a
nitrogen atmosphere. The solution was stirred at 0 ℃ for 30 min, followed by the addition of
EDC•HCl (1.15 g, 6 mmol) in portions. The solution was stirred at room temperature under
nitrogen atmosphere overnight. The mixture was diluted with CH2Cl2 (50 mL), washed with water
(30 mL), followed by brine (50 mL), concentrated under reduced pressure, and purified by column
chromatography (Hexane : EA = 1:1, v/v) to afford the desired compound as a yellow oil in a yield
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of 426 mg (62% NMR yield [determined from H integration and measured weight taking into
account residual DMAP]; Rf = 0.15 in EtOAc : hexane = 1:1, v/v) as a mixture with DMAP. It was
carried forward without further purification. 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.28 (t, J
= 8.0 Hz, 2H), 6.98 (t, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 6.34 (brs, 1H), 4.63 (s, 2H), 4.25
(t, J = 6.0 Hz, 2H), 3.29 (q, J = 6.3 Hz, 2H), 1.91-1.80 (m, 2H).
3-isocyanopropyl 2-phenoxyacetate. 3-formamidopropyl 2-phenoxyacetate (420 mg, 1.7 mmol)
was dissolved in dry DCM (3 mL), and dry pyridine (2 mL) was added. A solution of tosyl chloride
(TsCl, 646 mg, 3.4 mmol) in dry DCM (4 mL) was added to it dropwise at 0 ℃ under a nitrogen
atmosphere while stirring. The solution was stirred at room temperature under nitrogen atmosphere
overnight. The mixture was purified by column chromatography (Hexane : EA = 4:1, v/v) to afford
the desired compound as a brown oil in a yield of 191 mg (49%; Rf = 0.3 in EtOAc : hexane = 4:1,
v/v). 1H NMR (500 MHz, DMSO-d6) δ 7.27 (t, J = 7.6 Hz, 2H), 6.95 (t, J = 7.2, 1H), 6.88 (d, J =
8.0 Hz, 2H), 4.74 (s, 2H), 4.18 (t, J = 5.8 Hz, 2H), 3.50-3.41 (m, 2H), 1.94-1.84 (m, 2H). 13C{1H}
NMR (125 MHz, DMSO-d6) δ 168.7, 157.5, 156.1, 129.5, 121.2, 114.4, 64.4, 61.1, 38.0, 27.7.
HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C12H14NO3 220.0973; Found 220.0973.
1-isocyano-2-(2-(2-methoxyethoxy)ethoxy)ethane. Ethyl formate (67 mg, 0.9 mmol) was added
in portions to 2-(2-(2-methoxyethoxy)ethoxy)ethanamine (98 mg, 0.6 mmol) while stirring at 0 ℃.
The solution was removed from the ice-bath and was heated at 50 oC for 2 h. Excess ethyl formate
was removed by rotary evaporation to afford the desired product as a colorless oil in a near-
quantitative yield (>95%). This compound decomposed upon storage and was immediately used
in the next step. The intermediate was dissolved in dry DCM (2 mL), and dry pyridine (1 mL) was
added to it. A solution of TsCl (228 mg, 1.2 mmol) in dry DCM (2 mL) was added to it dropwise
at 0 ℃ under a nitrogen atmosphere while stirring. The solution was stirred at room temperature
under nitrogen atmosphere overnight. The mixture was purified by column chromatography
(Hexane : EA = 4:1, v/v) to afford the desired compound as a light brown oil in a yield of 68 mg
(63%; Rf = 0.25 in EtOAc : hexane = 4:1, v/v). 1H NMR (500 MHz, CDCl3) δ 3.72-3.61 (m, 8H),
3.58-3.51 (m, 4H), 3.36 (s, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 157.3, 72.0, 70.9, 70.7, 68.7,
59.1, 50.7, 41.8, 29.7. HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C8H16NO3 174.1130; Found
174.1123.
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The Supporting Information is available free of charge on the ACS Publications website at XXXX
Supporting figures and tables
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NMR spectra of products and intermediates
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Acknowledgements
R.M.F. gratefully acknowledges financial support from the University of Utah (VPR Seed grant),
the American Cancer Society (129785-IRG-16-190-01-IRG) the Huntsman Cancer Institute (GU
Center pilot grant), and the USTAR initiative. J.T. gratefully acknowledges financial support from
a University of Utah Skaggs Graduate Fellowship and from an American Foundation for
Pharmaceutical Education Pre-Doctoral Fellowship.
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