An efficient methodology to introduce o-(aminomethyl)phenyl-boronic acids into peptides: alkylation of secondary amines

Article abstract of DOI:10.1039/C6NJ02862D

Current approaches for incorporating boronic acids into peptides require one of the following: the synthesis of commercially unavailable pinacol-protected boronate ester amino acid building blocks, amidation of small-molecule amine-containing boronic acids, or reductive amination of amine residues with 2-formylphenyl boronic acid. These methods have drawbacks, such as the use of excess starting materials, the lack of reactive-site specificity, or the inability to add multiple boronic acids in solution. In addition, several of these approaches do not allow for incorporation of the critical o-aminomethyl functionality that allows for binding of saccharides under physiological conditions. In this work, we report three methods to functionalize synthetic peptides with boronic acids using solid-phase and solution-phase chemistries by alkylating a secondary amine with o-(bromomethyl)phenylboronic acid. Solution-phase chemistries afforded the highest yields, and were used to synthesize seven complex biotinylated multi-boronic acid peptides.

Full text of DOI:10.1039/C6NJ02862D

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An efficient methodology to introduce
o-(aminomethyl)phenyl-boronic acids into
peptides: alkylation of secondary amines†
Cite this:DOI: 10.1039/c6nj02862d
Erik T. Hernandez, Igor V. Kolesnichenko, James F. Reuther and Eric V. Anslyn*
Current approaches for incorporating boronic acids into peptides require one of the following: the
synthesis of commercially unavailable pinacol-protected boronate ester amino acid building blocks,
amidation of small-molecule amine-containing boronic acids, or reductive amination of amine residues
with 2-formylphenyl boronic acid. These methods have drawbacks, such as the use of excess starting
materials, the lack of reactive-site specificity, or the inability to add multiple boronic acids in solution.
In addition, several of these approaches do not allow for incorporation of the critical o-aminomethyl
functionality that allows for binding of saccharides under physiological conditions. In this work, we report
three methods to functionalize synthetic peptides with boronic acids using solid-phase and solution-phase
chemistries by alkylating a secondary amine with o-(bromomethyl)phenylboronic acid. Solution-phase
chemistries afforded the highest yields, and were used to synthesize seven complex biotinylated
multi-boronic acid peptides.
Received (in Montpellier, France)
12th September 2016,
Accepted 16th November 2016
DOI: 10.1039/c6nj02862d
www.rsc.org/njc
Introduction
Boronic acid containing compounds continue to gain interest in
the areas of chemosensing, materials chemistry, fluorescence
imaging, mass spectrometry, and biomedical engineering.1–24
From the sensing of sugars to the formation of dynamic covalent
structures, or serving as probes for the enrichment of analytes,
boronic acid compounds are seen as an important organic
moiety for developing novel analytical and in vivo applications.
Continued growth in the biological sciences has sparked interest
for synthesizing boronic acid containing peptides. These are
attractive synthetic targets because peptides are versatile plat-
Fig. 1 (a) Synthesized building block. Requires a total of five synthetic steps.
(b) Boronate ester moieties introduced at the C-terminus via coupling
conditions. (c) Polypeptide with ortho-aminomethyl boronic acid, synthe-
sized by reductive amination of 2-formylphenyl boronic acid.27–35
forms for incorporating binding specificity to a target of interest. The most common approach is incorporating a building block
Boronic acid peptides could also be used in the design of during solid-phase synthesis. However, long synthetic routes to
combinatorial libraries. A method for incorporating a boronic non-commercially available boronic acid reactants are required
acid into the side chains at controllable positions increases the (Fig. 1a).27–35 To derivatize the C-terminus, amidation of amino
diversity of compounds accessible. We believe these methods boronic acids is usually used (Fig. 1b). Recently, solid-phase
should be more modular; thus, providing greater utility when approaches for adding boronic acids at the C-terminus has been
compared to current approaches.25,26
reported.36 However, peptides with aspartates or glutamates have
Common approaches for synthesizing boronic acid containing to be avoided. If unprotected carboxylate side chains are used,
peptides include the use of building blocks with a boronate non specific amidation can occur. Such a limitation decreases the
ester side chain, amidation of boronic acid compounds, or diversity of peptides available by limiting the natural side chains
reductive amination of residues with 2-formylphenyl boronic acid. that can be used. Lastly, reductive amination, performed pre-
viously in our group, consisted of modifying only one amine
residue (Fig. 1c).30
Department of Chemistry, University of Texas at Austin, Austin, TX, 78712-1224,
Most boronic acids do not bind saccharides efficiently at
USA. E-mail: anslyn@austin.utexas.edu
neutral pH. While electron withdrawing groups on the phenyl
† Electronic supplementary information (ESI) available: NMR, HRMS, and HPLC
purity checks. See DOI: 10.1039/c6nj02862d
ring, such as in m-nitrophenylboronic acid, can act to promote
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boronate ester formation at neutral pH, the most common
structural feature that promotes binding is an o-aminomethyl
group.37,38 This structural feature is often overlooked when
incorporating a boronic acid into a receptor meant to be used
in a biological setting. The only previously published strategy
that allows for easy incorporation of an o-aminomethyl group
is reductive amination. Alternatively, it would be feasible to
incorporate the o-aminomethyl moiety into an amino acid
building block in advance of peptide synthesis, but this would
further increase the number of synthetic steps. The methods we
present in this paper were devised to be synthetically simple
and afford the critical o-aminomethyl group.
To increase the number of targetable sites on peptides while
minimizing the difficulty and time required to synthesize
o-(aminomethyl)phenylboronic acid building blocks, we developed
chemistries for functionalizing peptides with secondary amines.
Primary amines are present in the side chains of natural amino
acids such as lysine. However, alkylation of primary amines
typically suffers from over-alkylation. If secondary amines residues
are present, then alkylation approaches become a viable alterna-
tive. The commercial availability of o-(bromomethyl)phenylboronic
acid is an additional benefit to an alkylation approach.
Herein, we report three methods that introduce secondary
amines and subsequent o-(aminomethyl)phenylboronic acids to
peptides. The methods include both solid-phase and solution-
phase approaches. The first approach was the selective modifi-
cation of cysteine residues in solution using an iodoacetamide
linker with a Boc-protected secondary amine. In the second
approach, a cysteamine resin and an azido lysine building
block was used in the solid-phase synthesis of peptides with a
secondary amine. The third approach, using the commercially
available Ne-methyl lysine, was the most direct way for making
our desired peptides. Once a secondary amine was incorporated,
a reaction with o-(bromomethyl)phenylboronic acid afforded the
o-aminomethyl moiety on all our peptides.
Scheme 1 Synthesis of compound 1.
deprotection of the boronic ester were considered to be too
harsh for peptides, and thus unprotected boronic acids were
chosen. Irrespective of the low yield of this model reaction, we
pushed forward with peptide derivatization because of the ease
of the technique. The desired product was confirmed by mass
spectrometry and NMR.
After successful functionalization of DMEDA, as a proof of
concept experiment, we set out to devise a linker approach
to place secondary amines on peptides. Cysteine residues are
reported to be selectively modified with iodoacetamides. There-
fore, a linker with this functional tether provided a method for
selectively introducing a secondary amine.40 The synthesis of this
linker involved a single Boc protection of DMEDA (Scheme 2).
Literature accounts regularly describe mono-protection with
diamine compounds by using an excess of the amino com-
pound relative to Boc-anhydride.41 A mixture of products was
observed: unreacted starting material, singly protected product,
and doubly protected product. Aqueous washes removed unreacted
starting material. No further purification was performed because
the doubly protected product was inert in the following step.
Mono-Boc DMEDA was modified with chloroacetyl chloride, and
the desired product was isolated. A Finklestein reaction afforded
iodoacetamide linker 2. During purification, the doubly Boc
Finally, to demonstrate the usefulness of our chemistry for
making complex structures, seven peptides containing Ne-methyl
lysine amino acids and derivatized at the N-terminus with a
pegylated biotin, were synthesized. This organic moiety was seen
to add to the utility of boronic acid peptides for applications that
use streptavidin to be reported in future publications.
Scheme 2 Synthesis of mono-Boc diamine.
Results and discussion
Solution-phase incorporation of secondary amines and
alkylation
To first test if an alkylation strategy would be successful,
N,N0-dimethylethylenediamine (DMEDA) was used as a model
(Scheme 1). The alkylation occurred with a purified yield of
16% (1). The boronic acid community, has reported on the
difficulty to isolate boronic acid compounds because of the
strong interaction with the stationary phase in chromatographic
methods.39 Therefore, pinacol-protected boronate esters are
more commonly used due to facilitate the isolation of boronic
Scheme 3 Synthesis of compound 3. (a) MeCN, MeOH, TEA, Pyr, H2O, RT,
acid compounds. However, the reaction conditions required for 4 h. (b) TFA, TIS, H2O, RT, 4 h. (c) MeCN, H2O, RT, overnight.
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product was removed. The cysteine in Fmoc-CysAla-OH was o-(bromomethyl)phenylboronic acid (BMPBA) to give compound 3.
modified with the linker (Scheme 3). Deprotection with TFA Linker 2 should prove widely useful to selectively modify peptides
solution occurred quantitatively, followed by alkylation with with cysteine residues.31
Solid-phase incorporation of secondary amines and alkylation
To expand the utility of the alkylation approach, we sought to
develop solid-phase reactions for the incorporation of secondary
amines. Click approaches via Sharpless reactions, generally are
not compatible with boronic acids.42 Despite efforts to use
fluoride to minimize deborylation,43 copper clicking of boronic
acids did not work. A cysteamine resin and an azido lysine
building block also allowed incorporation of a secondary amine
(Scheme 4). The unnatural azido residue can be modified via
click chemistry while immobilized on the solid support.44 We
used N-methyl propargylamine (NMPA) to click on a secondary
amine. If the Fmoc protecting group is needed in future solution-
phase modification, deprotection by the basic amine was avoided
by doing repetitions of the click reaction. Three one-hour repeti-
tions were the maximum number before Fmoc deprotection of
product became significant.
Scheme 4 Synthesis of compound 4. (a) DMF, CuI, TBTA, sodium ascorbate,
RT, 1 h (3 repetitions). (b) MeCN, H2O, overnight. (c) TFA, TIS, H2O, RT, 4 h.
Scheme 5 Synthesis of compound 6. (a) DMF, DIPEA, RT, 2 h (3 repetitions). (b) TFA, TIS, H2O, RT, 4 h. (c) MeCN, H2O, RT, overnight.
Fig. 2 Biotinylated, using D-biotin, boronic acid peptides.
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Interestingly, Wang resin did not yield the desired product.
However, ‘‘clicking’’ did occur with a cysteamine resin. Upon
cleavage, the product (4) contained a thiol functional group as the
C-terminus, which could be used for solution-phase derivatiza-
tion if desired.
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Table 1 Biotinylated peptide library sequences and yields. PEG4-biotin was
placed on the N-terminus. X2 = –CH2CH2CH2CH2(NH(CH3)(CH2C6H6B(OH)2))
Compound
Sequence
Yield (%)
6
7
8
9
10
11
12
RTRX2LX2FX2Y
RTRX2LX2FGY
RTRX2LGFX2Y
RTRX2LGFGY
GTGX2LX2FX2Y
RTLX2RX2FX2Y
RTFX2LX2RX2Y
7.5
11
19
45
25
17
22
Building-block incorporation of secondary amines and
alkylation
Using commercially available and synthetically accessible Ne-methyl
lysine as a building block proved to be the most straightforward
method for introducing secondary amines. This building block
ensured the placement of multiple secondary amines along a
sequence with high reliability. Fmoc-Lys(Ne,Me)Ala-OH gave the
highest purified yield (60%; Scheme 5). Only a single modification
step post-solid phase synthesis was required to make a boronic acid
peptide. Thus, it minimized the cumulative inefficiencies at each
reaction step in the former two approaches. Using this approach,
seven complex biotinylated, using D-biotin, multi-boronic acid
peptides were synthesized (Scheme 5 and Fig. 2).
Of the seven biotinylated peptides, 9 had the highest purified
yield (45%). The purified yields of peptides with two or three
boronic acids ranged from 8–22%. We attributed the lower yields
to the increasing difficulty in purification when the number of
boronic acids grew. Streaking of product occurred more readily
during purification.
the commercially available Ne-methyl lysine building block was
used as the most direct method for making boronic acid peptides.
It gave the highest yield and was the method of choice to make a
set of seven biotinylated peptides. These peptides had up to three
boronic acids, although purification became difficult and the
associated yields decreased with increasing numbers of boronic
acids (Table 1).
Compounds such as 6–12 are currently being investigated as
pull-down reagents for cell-surface saccharides. Due to the utility
of the synthetic methodologies we report herein, we anticipate that
others can adopt them for site-specific incorporation of boronic
acids in peptides and proteins.
Experimental
The HPLC analytical trace for 9 gave a relatively sharp peak
as the product eluted. However, when two or three boronic side
chains were present, the elution peak was broadened, suggesting
again that the phenyl boronic acid moieties interact with the
column. Higher percentages of organic solvent were required to
elute the peptide. The broadening of the elution peak led to low
signal intensity during HPLC purification. This broadening led
to a low signal intensity during HPLC purification, which made
identification of the peaks challenging, and hence poor detec-
tion of sample elution was seen to contribute to the lower yields
for peptides with more than one boronic acid.
General methods
For automated Fmoc amino solid-phase peptide synthesis, Ala,
Pbf(Arg), Trt(Cys), Gly, Leu, Boc(Lys), Phe, and Boc(Thr) were
purchased from P3 biosystems. Fmoc-Lys(Ne,Me)-OH was pur-
chased from Chem. Pep. Inc. Fmoc-Lys(N3)-OH was purchased
from Chem-Impex, Inc. Cysteamine 2-ClTrt (0.95 mmol gꢀ1) resin
and Fmoc-Tyr(tBu)-Wang resin (0.46 mmol gꢀ1) were purchased
from AnaSpec, Inc. Fmoc-Ala-Wang (100–200 mesh, 0.72 mmol gꢀ1
)
was purchased NovaBiochem. DMF, DCM, piperidine used for
automated solid-phase peptide synthesis were purchased from
Fisher Scientific and Sigma-Aldrich. N,N0-Dimethylethylene-
diamine, chloroacetyl chloride, and sodium iodide, N-methyl
propargyl amine, copper iodide, and sodium ascorbate were
purchased from Sigma-Aldrich. o-(Bromomethyl)phenylboronic
Conclusions
We developed three different approaches for derivatizing pep- acid was purchased from Combi-Blocks. EZ-Linkt NHS-PEG4-
tides with secondary amines, followed by modification with biotin was purchased from Thermo Scientific.
o-(bromomethyl)phenylboronic acid. Unlike most previously
A Prelude peptide synthesizer (Protein Technologies, Inc.)
published methods for incorporation of boronic acids into was used for automated-solid phase synthesis of the peptides.
peptides, this alkylation strategy allows for easy incorporation For the longer peptides, a Liberty Blue microwave peptide
of an o-aminomethyl functionality on the aryl boronic acid, synthesizer was used. Preparative HPLC purification of pep-
which has been previously shown to increase binding affinity of tides was performed using an Agilent Zorbax SB-C18 Prep HT
boronic acids to saccharides. The first approach involved the column 21.2 ꢁ 250 mm; 10 mL minꢀ1, 5–95% MeOH in 90 min.
synthesis of a linker with an iodoacetamide at one side and Boc Analytical HPLC characterization of peptides was performed
protected secondary amine on the other end. Cysteine residues using an Agilent Zorbax column 4.6 ꢁ 250 mm; 1 mL minꢀ1
,
were selectively targeted in a model peptide. After deprotection, 5–95% MeCN (0.1% TFA) in 35 min (RT). A Gemini C18
the secondary amine was alkylated with a boronic acid. In the 3.5 micron 2.1 ꢁ 50 mm was used for online separation;
second approach, N-methyl propargyl amine was used in click 0.7 mL minꢀ1, 5–95% MeCN (0.1% formic acid) in 12 min
chemistry to introduce secondary amines followed by alkylation (RT). An Agilent Technologies 6530 Accurate Mass QTofLC/MS
with a boronic acid in the solid phase. Post cleavage, a boronic was used for high-resolution mass spectra of purified pep-
acid peptide with a C-terminal thiol group was isolated. Lastly, tides. Solvents used were HPLC grade. For small molecule
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organic compounds, reverse-phase combi-flash was used for and nanopure water (95 : 2.5 : 2.5) (4 h), followed by removal
purification if necessary.
of TFA, and precipitated with diethyl ether at 0 1C. No further
purification of the crude peptide was performed.
General procedure (A): synthesis of dimeric peptides and
Ne-methyl lysine peptides
Synthesis of compounds
Fmoc-CysAla-Wang resin, Fmoc-Lys(N3)Ala-S-resin, Fmoc-Lys(Ne,Me)-
(((Ethane-1,2-diylbis(methylazanediyl))bis(methylene))bis(2,1-
Ala-Wang, and biotinylated Ne-methyl lysine peptides resin phenylene))diboronic acid (1). N,N0-Dimethylethylenediamine
(100 mmol) were synthesized by automated sequential coupling (2.0 mmol) was dissolved in a solution of MeCN, MeOH, DMF
of Na-Fmoc-amino acid (0.1 M) in DMF in the presence of (1.4 mL, 71/14/14 (v/v/v)), followed by addition of 2-(bromo-
N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluoro- methyl)phenylboronic (4.0 mmol). The reaction was stirred overnight
phosphate (HBTU, 0.15 M) and Hu¨nig’s base (0.2 M) with gentle at RT. The compound was purified via reverse-phase combi-
nitrogen bubbling for 30 minutes at room temperature. A total of flash. Purified yield: 16%. HRMS-ESI+ (MeOH/H2O): calcd:
three repetitions were performed for each amino acid building m/z = 319.2031; found: m/z = 319.2006 [M ꢀ 2H2O + H]+.
block incorporated, followed by DMF (3 mL, 3 min, 3ꢁ) and 1H NMR (400 MHz, acetonitrile-d3) d 7.72 (dt, J = 7.4, 1.0 Hz, 1H),
DCM (3 mL, 3 min, 3ꢁ) washes. For peptides longer than three 7.66–7.63 (m, 1H), 7.61–7.58 (m, 1H), 7.38–7.23 (m, 6H), 5.00
amino acid residues, a 0.8 M LiCl wash (3 mL, 3 min, 3ꢁ) was (d, J = 0.8 Hz, 1H), 4.86 (s, 2H), 4.52 (s, 1H), 3.89 (s, 7H), 3.30
included after swelling with DCM. After the synthesis, resin was (d, J = 0.8 Hz, 2H), 2.78–2.70 (m, 1H).
washed with glacial AcOH (5 mL, 3ꢁ), DCM (5 mL, 3ꢁ), and
(2-(2-Iodo-N-methylacetamido)ethyl)(methyl)carbamate (2).
MeOH (5 mL, 3ꢁ). Resins were placed under vacuum overnight. Boc-anhydride (0.011 mol) was dissolved in 20 mL of DCM and
Fmoc-Cys Ala was cleaved from the resin using trifluoroacetic added dropwise to a solution of N,N0-dimethylethylenediamine
acid (TFA), triisopropylsilane, 1,2-ethanedithiol (EDT), and nano- (0.034 mol) in 30 mL of DCM under N2. The reaction was stirred
pure water (94 : 1.0 : 2.5 : 2.5) (4 h) for Fmoc-CysAla-OH. TFA was overnight at RT. The DCM was removed via rotary evaporation.
evaporated and the remaining oil was precipitated with diethyl The residual oil was washed with EtOAc, with water, and brine.
ether at 0 1C. No further purification of the crude peptide was Ethyl acetate was evaporated and a colorless oil remained. No
performed.
further purification was performed. The crude product (1 g) was
dissolved in 20 mL of dry DCM and placed in an ice bath, and
chloroacetyl chloride (0.0058 mmol) was introduced slowly
under N2. The solution turned dark brown. The reaction was
General procedure (B): solution-phase alkylation with
o-(bromomethyl)phenylboronic acid
Peptides were dissolved in a mixture of H2O and MeCN (0.6 mL, allowed to come to RT and stirred overnight. DCM was removed
1 : 1 v/v). To this solution, 0.1 mL of Hu¨nig’s base was added. via rotary evaporation. The remaining oil was dissolved in EtOAc
If the peptide precipitated, 0.15 mL of MeOH was added. The and washed with distilled water and brine (3ꢁ). The organic
solution was further diluted with 0.2 mL of MeCN, followed by layer was dried with Na2SO4. After filtration, EtOAc was removed
addition of 3.5 equivalents of o-(bromomethyl)phenylboronic by rotary evaporation. The compound was purified by normal-
acid. The reaction was allowed to stir overnight at RT. The following phase combi-flash (SI-1). Isolated product (0.11 mol) was dis-
day, additional boronic acid (3.5 equivalents) was added, followed solved in 1 mL of acetone followed by the introduction of NaI
by addition of 0.05 mL of Hu¨nig’s base. The reaction was incubated (0.22 mol). Precipitate formed after ten minutes. The reaction
at room temperature for an additional 2 h. Preparative HPLC was was stirred overnight at RT. The acetone was removed via rotary
used to purify the peptides. Purified samples were placed on the evaporation, followed by three washes with brine. No further puri-
rotary evaporator to remove MeOH. The aqueous remnants were fication was required. Yield 73%. LRMS-ESI/APC+/ꢀ (MeOH/H2O):
frozen and lyophilized overnight.
calcd: m/z = 379.2; found: m/z = 379.05 [M + Na]+.
Fmoc-C(B)A (3). Fmoc-CA-OH (0.07 mmol) was dissolved in
0.3 mL of H2O, followed by addition of 0.1 mL of MeOH : TEA :
Pyr : H2O (7 : 1 : 1 : 1) (v/v/v). A solution of compound 3 (0.07 mmol)
in 0.5 mL MeCN : H2O (69 : 31) (v/v) was introduced. The Boc
General procedure (C): solid-phase modification of the
N-terminus with EZ-Linkt NHS-PEG4-biotin: post automated
solid phase synthesis
Fmoc group was removed with 3 mL of piperidine (20% vol protecting group was removed using trifluoroacetic acid (TFA),
in DMF, 3 min, 3ꢁ) followed by washes with DMF (3 mL, 3 min, triisopropylsilane, and nanopure water (95 :2.5: 2.5) (4 h). TFA
3ꢁ), DCM (3 mL, 3 min, 3ꢁ), and 0.8 M LiCl (3 mL, 3 min, 3ꢁ). was removed, and the product was precipitated with diethyl ether
A 1.5 mL solution of EZ-Linkt NHS-PEG4-biotin was introduced at 0 1C. No further purification of the peptide was performed.
with 0.5 mL solution of 1.2 M Hu¨nig’s base. Gentle stirring The peptide was alkylated with o-(bromomethyl)phenylboronic
under nitrogen was performed for 1 h at RT, followed by washes acid following general procedure B. The peptide was purified
with DMF (3 mL, 3 min, 3ꢁ), DCM (3 mL, 3 min, 3ꢁ), and 0.8 M via preparative HPLC. MeOH was removed by rotary evapora-
LiCl (3 mL, 3 min, 3ꢁ). Coupling of biotin to peptide was tion, followed by freezing and lyophilization of aqueous
repeated a total of three times. Resins were washed with glacial remnants. Purified yield: 6%. HRMS-ESI+ (MeOH/H2O): calcd:
AcOH (5 mL, 3ꢁ), DCM (5 mL, 3ꢁ), and MeOH (5 mL, 3ꢁ), m/z = 659.2711; found: m/z = 659.2712 [M ꢀ 2H2O + H]+.
and resins were placed under vacuum overnight. Peptides 1H NMR (500 MHz, DMSO-d6) d 8.30 (s, 1H), 7.82 (d, J = 7.6 Hz,
were cleaved with trifluoroacetic acid (TFA), triisopropylsilane, 2H), 7.65 (t, J = 6.8 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.33–7.25 (m, 3H),
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7.16 (d, J = 7.3 Hz, 1H), 4.25 (t, J = 6.6 Hz, 2H), 4.20–4.11 (m, 1H), 4.28–4.15 (m, 3H), 4.04 (q, J = 7.2 Hz, 1H), 3.93 (dd, J = 9.0,
3.94 (d, J = 7.0 Hz, 1H), 3.88 (s, 4H), 3.72 (d, J = 10.9 Hz, 1H), 5.1 Hz, 1H), 3.66 (q, J = 7.3 Hz, 2H), 3.50–3.45 (m, 1H), 3.43–3.37
3.57–3.39 (m, 2H), 3.40–3.30 (m, 1H), 2.91 (dd, J = 13.8, 5.2 Hz, (m, 1H), 2.19–2.12 (m, 3H), 1.61 (dt, J = 13.8, 7.0 Hz, 1H), 1.49
1H), 2.71 (d, J = 14.0 Hz, 3H), 2.68–2.58 (m, 4H), 2.24 (s, 2H), (h, J = 7.6 Hz, 2H), 1.20 (dd, J = 9.4, 7.1 Hz, 4H).
2.16 (s, 1H), 1.18 (dd, J = 7.0, 1.7 Hz, 3H). 13C NMR (126 MHz,
Biotin-ArgThrArgLys(Ne,Me,B)LeuLys(Ne,Me,B)PheLys(Ne,Me,B)-
DMSO-d6) d 170.31, 156.90, 144.37, 144.36, 144.25, 141.36, Tyr-OH (6). General procedure B was used for 0.022 mmol of
135.41, 135.40, 130.34, 130.14, 128.60, 128.02, 126.00, 120.83, starting material. Purified yield: 7.5%. HRMS-ESI+ (MeOH/H2O):
66.76, 63.25, 63.23, 54.97, 54.97, 52.75, 50.19, 47.26, 47.26, 44.12, calcd: 701.0625; found: m/z = 701.0638 [M ꢀ 3H2O + 3H]3. 1H NMR
41.44, 40.45, 40.45, 40.06, 40.02, 39.90, 39.87, 39.84, 39.73, 39.70, (500 MHz, DMSO-d6) d 8.29 (s, 8H), 7.65–7.50 (m, 3H), 7.33–7.03
39.67, 39.56, 39.53, 39.51, 39.40, 39.37, 39.34, 39.23, 39.20, 39.17, (m, 18H), 6.93 (d, J = 7.8 Hz, 3H), 6.58 (d, J = 7.9 Hz, 3H), 4.51–4.30
39.04, 39.01, 35.87, 34.48, 34.12, 18.92, 18.91.
(m, 2H), 3.57 (q, J = 8.5, 7.5 Hz, 5H), 3.46 (d, J = 8.9 Hz, 9H), 3.37
Fmoc-Lys(N3,B)Ala-SH (4). Fmoc-Lys(N3)Ala-S-resin. Mixing (t, J = 5.7 Hz, 2H), 3.24 (s, 1H), 3.20–3.11 (m, 2H), 3.04 (dt, J =
of solvents, preparation of click solutions, and click reaction 26.2, 7.3 Hz, 6H), 2.91 (s, 5H), 2.83–2.63 (m, 7H), 2.42–2.24 (m,
was done under N2. Resin (100 mmol) and alkyne (1 eq.) were 10H), 2.23–2.00 (m, 12H), 1.70–1.32 (m, 29H), 1.29–1.05 (m, 14H),
combined with 1 mL of DMF followed by the addition of 0.2 mL 1.04–0.90 (m, 3H), 0.78 (dtd, J = 33.8, 18.2, 17.3, 5.5 Hz, 9H).
solution of CuI (0.5 eq.), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)-
Biotin-ArgThrArgLys(Ne,Me,B)LeuLys(Ne,Me,B)PheGlyTyr-OH
methyl]amine (TBTA) (1 eq.), and sodium ascorbate (1 eq.) in (7). General procedure B was used for 0.021 mmol of starting
DMF and stirred for 1 h at RT. The reaction solution was material. Purified yield: 11%. HRMS-ESI+ (MeOH/H2O): calcd:
drained from the resin and fresh reaction solution was intro- m/z = 951.0221; found: m/z = 951.0258 [M ꢀ 3H2O + 2H]2+
.
duced two more times. The resin was washed with DMF (3 mL, HRMS-ESIꢀ (MeOH/H2O): calcd: m/z = 949.0075; found: m/z =
1
3 min, 3ꢁ), DCM (3 mL, 3 min, 3ꢁ), glacial AcOH (5 mL, 3ꢁ), 949.0051 [M ꢀ 2H2O ꢀ 2H]2ꢀ. H NMR (499 MHz, DMSO-d6)
DCM (5 mL, 3ꢁ), and MeOH (5 mL, 3ꢁ). The resin was then d 8.35 (s, 8H), 8.16 (s, 4H), 7.59 (s, 3H), 7.20–7.05 (m, 4H), 6.94
placed under vacuum overnight, followed by cleavage using (q, J = 8.8 Hz, 2H), 6.54 (d, J = 8.3 Hz, 1H), 4.34–4.27 (m, 1H),
trifluoroacetic acid (TFA), triisopropylsilane, 1,2-ethanedithiol 4.13 (dd, J = 7.9, 4.3 Hz, 1H), 3.42–3.35 (m, 3H), 2.91 (s, 1H),
(EDT), and nanopure water (94 : 1.0 : 2.5 : 2.5) (4 h). TFA was 2.81–2.65 (m, 1H), 2.57 (d, J = 12.6 Hz, 2H), 2.45–2.17 (m, 1H),
evaporated and remaining oil was precipitated with diethyl ether 2.12–1.99 (m, 13H), 1.69 (d, J = 17.0 Hz, 1H), 1.63–1.55 (m, 1H),
at 0 1C. Peptide was purified via preparative HPLC. MeOH was 1.54–1.34 (m, 18H), 1.33–1.22 (m, 1H), 1.18 (d, J = 7.1 Hz, 5H),
removed by rotary evaporation, followed by freezing and lyophi- 1.00 (td, J = 14.0, 12.8, 6.7 Hz, 4H), 0.86 (d, J = 6.1 Hz, 1H),
lization of aqueous remnants. Purified yield: 14%. HRMS-ESI+ 0.80 (t, J = 5.6 Hz, 4H), 0.74 (dd, J = 10.5, 4.6 Hz, 1H). 13C NMR
(MeOH/H2O): calcd: m/z = 750.3222; found: m/z = 750.3228 (126 MHz, DMSO-d6) d 142.07, 133.86, 129.20, 129.17, 127.81,
[M + Na]+. 1H NMR (500 MHz, DMSO-d6) d 8.30 (s, 1H), 7.97 127.04, 74.53, 57.89.
(d, J = 6.7 Hz, 1H), 7.84 (t, J = 6.8 Hz, 2H), 7.65 (q, J = 6.2 Hz, 2H),
Biotin-ArgThrArgLys(Ne,Me,B)LeuGlyPheLys(Ne,Me,B)Tyr-OH
7.58 (d, J = 7.1 Hz, 1H), 7.39 (q, J = 7.0 Hz, 2H), 7.29 (q, J = 7.5, (8). General procedure B was used for 0.015 mmol of starting
7.0 Hz, 2H), 7.22 (dd, J = 8.7, 6.6 Hz, 1H), 7.15 (q, J = 6.8 Hz, 1H), material. Purified yield: 19%. HRMS-ESI+ (MeOH/H2O): calcd:
4.30 (t, J = 6.8 Hz, 2H), 4.22 (dd, J = 7.4, 4.0 Hz, 2H), 4.16 (td, J = m/z = 634.6855; found: m/z = 634.6832 [M ꢀ 2H2O + 3H]3+.
6.9, 3.3 Hz, 2H), 3.93 (dd, J = 9.4, 4.9 Hz, 1H), 3.65 (d, J = 4.4 Hz, 1H NMR (500 MHz, DMSO-d6) d 8.34 (s, 9H), 7.84 (t, J = 5.5 Hz,
2H), 3.47 (d, J = 5.2 Hz, 0H), 3.33 (dt, J = 13.5, 6.8 Hz, 1H), 3.25 1H), 7.66 (s, 1H), 7.37–6.87 (m, 3H), 6.58 (d, J = 8.4 Hz, 3H), 6.41
(dt, J = 13.4, 6.6 Hz, 1H), 2.78–2.63 (m, 2H), 2.03 (d, J = 4.5 Hz, (s, 1H), 6.36 (s, 1H), 4.32–4.28 (m, 1H), 4.12 (d, J = 7.2 Hz, 1H),
3H), 1.77 (d, J = 9.3 Hz, 3H), 1.64 (s, 1H), 1.52 (d, J = 11.8 Hz, 3.99 (d, J = 24.1 Hz, 1H), 3.21–3.14 (m, 2H), 3.10–2.89 (m, 1H),
1H), 1.35–1.19 (m, 4H), 1.16 (d, J = 7.1 Hz, 3H). 13C NMR 2.85–2.78 (m, 2H), 2.39–2.29 (m, 1H), 2.21 (q, J = 0.5 Hz, 2H),
(126 MHz, DMSO-d6) d 172.78, 172.08, 156.53, 144.13, 144.03, 1.63–1.35 (m, 6H), 1.33–1.09 (m, 3H), 1.01 (dd, J = 15.0, 6.0 Hz,
143.25, 141.86, 141.07, 134.48, 129.44, 129.21, 129.09, 128.16, 4H), 0.84 (ddd, J = 23.6, 11.2, 5.7 Hz, 8H). 13C NMR (126 MHz,
127.57, 127.19, 125.65, 124.80, 121.78, 120.51, 72.44, 66.14, DMSO-d6) d 179.31, 172.13, 165.36, 162.71, 157.24, 129.20,
61.71, 60.55, 54.72, 49.83, 49.70, 48.63, 47.02, 42.26, 40.28, 127.96, 69.75, 63.36, 61.04, 59.20, 55.40, 39.12, 35.09, 30.67,
40.11, 40.02, 39.94, 39.86, 39.77, 39.69, 39.60, 39.52, 39.43, 30.65, 28.17, 28.02, 25.24.
39.35, 39.26, 39.18, 39.07, 39.02, 38.16, 37.41, 35.91, 31.38,
29.67, 22.76, 18.51.
Biotin-ArgThrArgLys(Ne,Me,B)LeuGlyPheGlyTyr-OH (9). General
procedure B was used for 0.015 mmol of starting material. Purified
Fmoc-Lys(Ne,Me,B)A-OH (5). General procedure B was used for yield: 45%. HRMS-ESI+ (MeOH/H2O): calcd: m/z = 567.6386; found:
0.054 mmol of starting material. Purified yield: 60%. Positive m/z = found 567.6403 [M ꢀ H2O + 3H]3+. HRMS-ESIꢀ (MeOH/H2O):
HRMS-ESI+ (MeOH/H2O): calcd: m/z = 610.27010; found: calcd: m/z = 848.9370; found: m/z = 848.9370 [M ꢀ H2O ꢀ 2H]2ꢀ
.
m/z = 610.2685 [M + Na]+. Negative HRMS-ESIꢀ (MeOH/H2O): 1H NMR (500 MHz, DMSO-d6) d 8.35 (s, 8H), 8.13 (s, 1H), 7.92–7.57
calcd: m/z = 586.2736; found: m/z = 586.2727 [M ꢀ H]ꢀ. 1H NMR (m, 2H), 7.55 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.34 (d,
(400 MHz, DMSO-d6) d 8.23 (s, 1H), 7.84 (dd, J = 7.6, 3.1 Hz, 2H), J = 5.8 Hz, 0H), 7.32–7.26 (m, 3H), 7.26–7.18 (m, 7H), 7.18–7.02
7.68–7.61 (m, 2H), 7.50 (dt, J = 7.1, 1.1 Hz, 1H), 7.39 (tt, J = 7.5, (m, 2H), 6.94 (d, J = 8.3 Hz, 3H), 6.59 (d, J = 7.9 Hz, 3H), 6.39
1.6 Hz, 2H), 7.35–7.30 (m, 2H), 7.30–7.26 (m, 1H), 7.23 (ddd, (d, J = 28.5 Hz, 2H), 4.88 (s, 1H), 4.76 (s, 1H), 4.64 (d, J = 4.2 Hz,
J = 7.2, 5.3, 3.4 Hz, 1H), 7.18 (d, J = 7.2 Hz, 1H), 4.63 (s, 2H), 1H), 4.58 (s, 2H), 4.44 (s, 0H), 4.36 (s, 1H), 4.32–4.27 (m, 1H),
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4.20 (s, 3H), 4.13 (s, 2H), 4.02 (s, 2H), 3.88–3.70 (m, 3H), 3.59 (d, 1.79–1.30 (m, 28H), 1.27–1.21 (m, 1H), 1.06–0.88 (m, 3H), 0.77 (dt,
J = 6.4 Hz, 2H), 3.49 (d, J = 2.6 Hz, 10H), 3.39 (t, J = 6.0 Hz, 2H), J = 27.5, 7.3 Hz, 8H). 13C NMR (126 MHz, DMSO-d6) d 235.31,
3.31 (s, 1H), 3.28 (d, J = 5.1 Hz, 5H), 3.22–3.15 (m, 3H), 3.04 (s, 175.67, 174.39, 173.56, 173.06, 172.72, 172.43, 171.79, 171.67,
6H), 2.94 (d, J = 12.8 Hz, 1H), 2.82 (dd, J = 12.5, 5.1 Hz, 2H), 2.74 171.22, 167.42, 164.16, 160.27, 160.01, 157.35, 157.30, 155.95,
(s, 2H), 2.58 (d, J = 12.4 Hz, 1H), 2.46–2.28 (m, 2H), 2.22–2.20 142.27, 138.02, 135.25, 134.31, 131.05, 130.22, 129.87, 129.81,
(m, 2H), 1.47 (d, J = 25.5 Hz, 20H), 1.35–1.16 (m, 2H), 1.02 (d, 129.19, 128.94, 128.54, 127.66, 127.24, 127.23, 118.79, 116.42,
J = 8.1 Hz, 4H), 0.83 (d, J = 22.7 Hz, 8H). 13C NMR (126 MHz, 115.59, 110.38, 74.96, 70.39, 70.37, 70.31, 70.18, 69.57, 67.28,
DMSO-d6) d 172.45, 172.13, 171.90, 171.66, 171.53, 171.15, 67.03, 62.00, 60.24, 60.13, 58.19, 56.29, 56.15, 54.43, 53.62,
170.33, 170.15, 168.71, 168.71, 167.56, 166.37, 162.73, 157.29, 52.39, 49.34, 42.70, 41.00, 40.48, 40.28, 40.17, 39.25, 37.84, 37.10,
157.29, 155.45, 143.52, 142.89, 142.27, 138.01, 137.98, 134.00, 36.54, 35.91, 31.41, 29.10, 29.08, 28.92, 28.67, 25.97, 25.43, 24.87,
133.99, 133.73, 133.25, 130.13, 129.09, 128.93, 128.56, 128.33, 24.21, 23.63, 23.47, 23.47, 23.34, 23.33, 22.07, 20.17.
128.05, 127.37, 126.95, 126.81, 126.19, 126.04, 125.72, 125.63,
Biotin-ArgThrPheLys(Ne,Me,B)LeuLys(Ne,Me,B)ArgLys(Ne,Me,B)-
125.35, 114.66, 73.89, 73.37, 73.33, 73.24, 73.20, 69.76, 69.70, Tyr-OH (12). General procedure B was used for 0.022 mmol
69.67, 69.55, 69.45, 69.15, 66.76, 66.34, 61.05, 59.22, 58.44, of starting material. Purified yield: 22%. Positive HRMS-ESI+
57.64, 57.59, 57.52, 57.49, 57.47, 55.88, 55.40, 51.87, 40.78, (MeOH/H2O): calcd: m/z = 701.3950; found: m/z = found 701.3975
40.34, 39.45, 39.13,38.45, 37.19, 35.85, 35.09, 30.97, 30.79, [M ꢀ H2O + 3H]3+. HRMS-ESIꢀ (MeOH/H2O): calcd: m/z = 1050.0735;
1
30.65, 30.47, 28.66, 28.18, 28.03, 25.25, 24.73, 24.12, 22.97, 21.60, found: m/z = 1050.0684 [M ꢀ 3H2O ꢀ 2H]2ꢀ. H NMR (500 MHz,
21.47, 20.08, 19.76, 18.69.
DMSO-d6) d 8.28 (s, 8H), 7.65–7.57 (m, 3H), 7.31–7.20 (m, 11H),
Biotin-GlyThrGlyLys(Ne,Me,B)LeuLys(Ne,Me,B)PheLys(Ne,Me,B)- 7.15 (t, J = 11.0 Hz, 16H), 6.90 (d, J = 8.1 Hz, 0H), 6.85 (d, J = 8.1 Hz,
Tyr-OH (10). General procedure B was used for 0.015 mmol of 2H), 6.62–6.50 (m, 3H), 6.37 (d, J = 19.1 Hz, 0H), 4.51 (s, 1H), 4.49
starting material. Purified yield: 25%. HRMS-ESI+ (MeOH/H2O): (s, 1H), 4.31 (dd, J = 7.8, 5.0 Hz, 2H), 4.29–4.22 (m, 0H), 4.22–4.09
calcd: m/z = 635.3418; found: m/z = 635.3437 [M ꢀ H2O + 3H]3+
.
(m, 2H), 3.98 (s, 2H), 3.65–3.51 (m, 4H), 3.50–3.46 (m, 12H), 3.44
HRMS-ESIꢀ (MeOH/H2O): calcd: m/z = 950.9938; found: m/z = (s, 4H), 3.37 (t, J = 5.8 Hz, 3H), 3.24 (d, J = 1.3 Hz, 1H), 3.16
1
949.9907 [M ꢀ 3H2O ꢀ 2H]2ꢀ. H NMR (500 MHz, DMSO-d6) (t, J = 5.9 Hz, 3H), 3.11–2.87 (m, 4H), 2.79 (dd, J = 12.6, 5.2 Hz, 3H),
d 8.30 (s, 6H), 7.64 (d, J = 7.2 Hz, 3H), 7.34–7.19 (m, 4H), 7.15 (d, 2.20–2.17 (m, 1H), 1.95–1.91 (m, 1H), 1.82–1.73 (m, 0H), 1.59 (s, 7H),
J = 8.8 Hz, 12H), 6.94 (d, J = 7.5 Hz, 3H), 6.59 (d, J = 7.8 Hz, 3H), 1.54–1.32 (m, 26H), 1.28 (p, J = 7.1 Hz, 1H), 1.18 (d, J = 18.2 Hz, 8H),
4.96 (s, 0H), 4.52 (d, J = 1.8 Hz, 1H), 4.49–4.39 (m, 1H), 4.31 (t, 0.95 (d, J = 6.3 Hz, 4H), 0.81 (dd, J = 22.7, 6.6 Hz, 9H).
J = 6.4 Hz, 1H), 4.13 (dd, J = 7.8, 4.3 Hz, 2H), 4.00 (t, J = 5.3 Hz,
1H), 3.38 (t, J = 5.9 Hz, 2H), 3.25 (d, J = 1.7 Hz, 1H), 3.20–3.13
(m, 2H), 3.11–2.98 (m, 1H), 2.93 (d, J = 9.7 Hz, 1H), 2.83–2.67
Acknowledgements
(m, 2H), 2.57 (d, J = 12.5 Hz, 1H), 2.50 (p, J = 1.8 Hz, 11H), 2.45–
We thank our following funding sources: the National Institute
2.31 (m, 6H), 2.30–2.21 (m, 0H), 2.21–2.17 (m, 1H), 1.94 (s, 1H),
of Health (5DP1GM106408), the Defense Advanced Research
1.66–1.56 (m, 1H), 1.55–1.33 (m, 13H), 1.27 (q, J = 7.8 Hz, 1H),
Projects Agency (DARPA, N66001-14-2-4051), and the Welch Regents
1.22 (dd, J = 6.6, 2.2 Hz, 9H), 1.12 (d, J = 19.8 Hz, 0H), 1.07–0.94
Chair (F-0046).
(m, 4H), 0.93–0.68 (m, 6H). 13C NMR (126 MHz, DMSO-d6)
d 234.84, 234.82, 180.21, 172.94, 171.36, 170.97, 169.88, 169.35,
165.77, 163.23, 155.70, 142.16, 141.70, 135.07, 133.88, 130.93,
130.55, 129.64, 129.64, 129.41, 129.07, 129.07, 128.30, 127.61,
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60.46, 59.50, 57.78, 55.79, 55.72, 53.45, 42.42, 41.85, 40.32, 40.14,
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