Exploiting azido-dichloro-triazine as a linker for regioselective incorporation of peptides through their N, O, S functional groups

Article abstract of DOI:10.1016/j.bioorg.2020.104334

In the field of bioconjugation, linker development has witnessed massive growth in recent years. 2,4,6-Trichloro-1,3,5-triazine (TCT) is a tridentate linker that can accommodate three distinct nucleophiles. Herein, the reaction of azido triazine derivativ

Full text of DOI:10.1016/j.bioorg.2020.104334

Journal Pre-proofs
Exploiting Azido-dichloro-Triazine as a Linker for Regioselective Incorpora-
tion of Peptides through their N, O, S Functional Groups
Anamika Sharma, Ashish Kumar, Ayman El-Faham, Beatriz G. de la Torre,
Fernando Albericio
PII:
S0045-2068(20)31632-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.104334
YBIOO 104334
Reference:
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Bioorganic Chemistry
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11 August 2020
16 September 2020
20 September 2020
Please cite this article as: A. Sharma, A. Kumar, A. El-Faham, B.G. de la Torre, F. Albericio, Exploiting Azido-
dichloro-Triazine as a Linker for Regioselective Incorporation of Peptides through their N, O, S Functional
Groups, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104334
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Exploiting Azido-dichloro-Triazine as a Linker for Regioselective Incorporation of
Peptides through their N, O, S Functional Groups
Anamika Sharma,1 Ashish Kumar,1,2 Ayman El-Faham,3,4 Beatriz G. de la Torre,1,2,*
Fernando Albericio1,3,5,6,*
1Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal, Durban
4001, South Africa
2KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), School of Laboratory Medicine
and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4041, South
Africa
3Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi
Arabia
4Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, 12321
Alexandria, Egypt
5CIBER-BBN (Networking Centre on Bioengineering, Biomaterials and Nanomedicine) and Department
of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain
6Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), 08034 Barcelona, Spain
Abstract
In the field of bioconjugation, linker development has witnessed massive growth in recent years.
2,4,6-Trichloro-1,3,5-triazine (TCT) is a tridentate linker that can accommodate three distinct
nucleophiles. Herein, the reaction of azido triazine derivatives with nucleophiles (amine, thiol and
phenol) is studied. The replacement of first chlorine was performed at 0 °C while that of the last
chlorine was achieved successfully at rt. As a proof of concept of this strategy with potential
application in biological studies, pentapeptides (Ac-XGGFL-NH2 where X = Lys or Tyr or Cys)
were reacted with 2-azido-4,6-dichlorotriazine to replace the first and second chlorine at 0 °C and
at rt, respectively. The reactivity of 2-azido-4,6-dichlorotriazine was found to be similar for the α
and ε amine group present in same peptide. These findings demonstrate the applicability of 2-
azido-4,6-dichlorotriazine as a linker with potential further application in bioconjugation.
1

Introduction
In the field of bioconjugation, LINKER development has witnessed massive growth in recent years
[1-4]. The linker is usually a spacer molecule that connects drugs to two or more biomolecules
(drugs, peptides or proteins) of biological significance [4-6]. It must ensure the stability of a new
molecule after bioconjugation without altering the biological significance of the biomolecules
attached to it and must facilitate the controlled release of the drug/peptide at the target site [6-8].
Due to its low cost and ease of sequential incorporation of three distinct nucleophiles at different
temperatures, 2,4,6-Trichloro-1,3,5-triazine (TCT) is frequently used as a building block in
organic synthesis to access complex molecular architectures [9-11]. The three reactive chlorines
present in TCT offers uniqueness, responsible for its efficient reactivity with nucleophiles. The
incorporation of the first nucleophile into TCT is carried out at 0 °C, the second at room
temperature (rt), and the third under heating or even reflux conditions, which cannot be considered
biologically compatible (Figure 1).
Cl
Nu1
Nu1
Nu1
0 oC
rt
> 90 oC
N
N
N
N
N
N
N
N
Cl
N
Cl
Cl
N
Cl
Nu2
N
Cl
Nu2
N
Nu3
Figure 1. Sequential incorporation of nucleophiles (Nu1, Nu2 and Nu3)
The loss of σ-bond electron withdrawal (of a chlorine atom) and the increase in π-orbital electron
density (electron-donating property of the incorporated nucleophile) on the triazine ring decreases
the reactivity for further nucleophilic substitution reactions, and hence elevated temperatures are
required for further reactions [9, 12-16]. In our earlier work, we demonstrated the concept of
orthogonal chemoselectivity, which was defined as “discrimination between reactive sites in any
order” using amine, thiol and alcohol/phenol to find the preferential order of incorporation onto
TCT as a linker [14, 17]. However, the last replacement was achieved at a higher temperature [14].
To address this, and given the electron-withdrawing behavior of azide functionality [18], in another
study we introduced azide as one of the nucleophiles, in addition to thiol, alcohol and phenol, onto
TCT. Azide incorporation onto TCT rendered the last chlorine replacement at rt, thus being
compatible with a biological context. In addition to the above, azide functionality also unfolds the
2

possibility of further transformation mainly via click chemistry to incorporate another chemical
entity [19].
The idea of not incorporating amine in our previous work, as once amine is incorporated it’s not
possible for any other nucleophile to react with TCT and hence posing a limitation for triorthogonal
chemoselectivity substitution. However, the amine-bearing triazine motif is the “Master Key” as
it is integral part of several commercial molecules [20]. Therefore, in the present study, we
examined the TCT core with three possible substituents, where one is occupied by azide and
another by amine. The third substituent could be any nucleophile viz., amine, thiol, alcohol or
phenol. Also, as an application in biological systems, we also report the reactivity of TCT using
peptides bearing Lys, Cys and Tyr, which contain amine, thiol and phenol nucleophiles,
respectively, and mimics biological systems.
Result and Discussion
The first target of the present study was to explore the possible combinations in the sequential
order of incorporating different nucleophiles to obtain the triazine core, where one substitution is
azide (facilitating all process and/or further modification through e.g. Click chemistry), another
one amine (due to its importance in biological systems) and the third alcohol or thiol or phenol or
again amine. Figure 2 shows the nucleophiles used in this part of the study. Through
NH2
OH
SH
3-methylbutan-1-amine
3-methylbutan-1-ol
3-methylbutane-1-thiol
OH
NaN3
Sodium azide
phenol
X
NH
N
N3
X= NH, O, S
O
XR=
N
N
XR
Figure 2. Nucleophiles chosen for synthesizing tri-substituted triazine. Through the whole text,
the following color code has been used: dark blue for azide, blue for amine, red for alcohol, pink
for phenol, and green for thiol
3

All the possible combinations to synthesize tri-substituted TCT, under the previous indicated
premises -N3 and amine substitution are shown in Scheme 1. Azide was evaluated at all three
positions. TCT-N3 (1) was synthesized as described earlier by our group [18]. For the second
incorporation, amine, thiol, alcohol and phenol were reacted with TCT-N3 at 0 ºC (-20 ºC for
phenol) for 30 mins. Successful incorporation was observed in case of amine, thiol and phenol,
affording the formation of 6, 7 and 8. However, alcohol yielded no product, as demonstrated earlier
[18]. The use of a lower temperature for the case of phenol is due to the deactivating effect of the
phenol, which once incorporated facilitates the incorporation of a second molecule [17].
The di-substituted derivatives (6, 7 and 8) were further treated with amine for the third substitution.
The reaction was performed at rt for 3 h (18 h in case of 6). Successful incorporation of amine
onto the di-substituted derivatives was observed in all cases affording 14, 15 and 16. The
incorporation of a second amine when an amine is already present (6) is possible but more
demanding than other cases.
4

NH
N
N
O
N
N3
NH
NH
16
N
N
N
N
Cl
S
N
N3
N
N
N3
N
N
H
NH
15
14
O
N
8
N3
Cl
N
N
N
N3
Cl
N
N
N
9
Cl
H
Cl
S
N
7
N3
N
N
Cl
N
N
6
N3
Cl
Cl
N
H
N
N
NH
N
N
N
N
N
N
2
Cl
H
N
N
Cl
N
N3
N
1
H
N
N
N3
6
H
14
Cl
N
N
N
O
N
O
O
Cl
Cl
N
N
O
N
N
N
N
N
TCT
Cl
N3
N
N
N
H
N
Cl
N
Cl
10
13
Cl
Cl
5
3
O
N
Cl
O
N
N
N
N
N
O
N
N
HN
N3
O
Cl
N
S
N
N
N
N3
H
4
N
N
17
HN
N
Cl
16
Cl
N
N3
11
8
Cl
N3
N
N
N
N
N
N
N
S
Cl
S
H
12
7
N3
N
N
N
N
S
H
15
Scheme 1. Nucleophilic substitution reactions carried out in this study.
To evaluate azide at the second position, mono-substituted TCTs (m-TCTs) (2, 3, 4 and 5) were
synthesized successfully using amine, alcohol, thiol, and phenol, respectively, and TCT at 0 ºC (-
20 ºC in case of phenol) for 30 mins. m-TCTs were reacted with sodium azide at 0 ºC for 30 mins
to form disubstituted TCT (d-TCT) derivatives (6, 10, 7 and 8, respectively) with full conversion
in all cases. d-TCTs were reacted further with amine at rt for 3 h for the third substitution. Full
conversion of 6, 7, and 8 to the tri-substituted TCT (14, 15 and 16, respectively) was observed.
However, the reaction of 10 (alcohol substituent) with amine at rt for 16 h rendered low conversion
as monitored by TLC. Then, the reaction was heated at 40 ºC for 16 h to completion, affording 17.
5

To evaluate azide substitution at the third position, m-TCTs (2, 3, 4 and 5) were reacted with amine
at rt for 2 h. All the reactions afforded d-TCTs (9, 11, 12 and 13, respectively). Finally, compounds
9, 11, 12 and 13 were dissolved in acetone and reacted with sodium azide in water at 40 ºC for 2
days. Product formation was only observed in 12 and 13, affording 15 and 16, respectively.
Summary of all these experiments are shown in Table 1.
Table 1: Reactivity of TCT with nucleophiles.
Order*
1st
Compound
Yield
(%)
2nd
3rd
14
15
16
17
91%
94%
91%
90%
91%
N3
NH
SH
pOH**
OH
N3
NH
NH
NH
NH
NH
N3
✓
N3
✓
N3
✓
N3
✓
NH
NH
SH
✓
NH
N3
n.r.
-
93%
NH
N3
✓
✓
93%
91%
90%
90%
-
SH
NH
N3
pOH
pOH
OH
OH
NH
N3
✓
✓
NH
N3
NH
N3
✓
NH
n.r.
*Indicates the order of nucleophile incorporation onto TCT; **pOH = Phenolic
✓ = product obtained; n.r. = no reaction
In addition to these reactions, a competitive experiment was conducted at each substitution level
to prove the selectivity of amine as nucleophile in the presence of other nucleophiles in the same
reaction mixture (Scheme 2, Table 2). Thus, TCT-N3 (1 equiv) was simultaneously treated with
amine, thiol, alcohol and phenol (1 eq. each) in one pot in order to shed light on the competition
between nucleophiles for the formation of product. The reaction was stirred at 0 ºC for 30 mins
6

and afforded only the amine derivative (6) as demonstrated by TLC and confirmed by HPLC. Even
phenol did not react with TCT-N3 in the presence of amine in the same reaction.
A competitive experiment was also conducted for each d-TCT, which afforded only amine
substituted derivatives (Table 2), as expected from our earlier results. The idea of a competitive
experiment is crucial in bioconjugation, as most peptides/antibodies have amino acids with side
chain functional groups. Here we demonstrate the selectivity of amine towards nucleophilic
substitution reaction onto TCT derivatives, affording only one product.
Cl
Cl
N
N
N
N
O
N
N
N3
N
N3
N
Cl
O
N
N
N
H
N
N
N3
Cl
N3
Cl
O
O
N
N
N
N
N
N
N3
N
SH
H
N
H
N3
NH2
OH
OH
1:1:1:1
one pot
NH
NH
S
N
S
N
N
N
N
N
N
N
N
N
N3
N
Cl
N3
N
N
H
N3
N3
N
Cl
H
Scheme 2. Competitive reaction carried out on TCT-N3 (1) and d-TCTs (6, 7, 8 and 10) with azide
functionality
Table 2: Competition experiments
Compound Position 1*
Position 2*
One pot addition
Product obtained
Only 6
2
N3
N3
N3
N3
N3
-
NH+SH+OH+pOH
NH+SH+OH+pOH
NH+SH+OH+pOH
NH+SH+OH+pOH
NH+SH+OH+pOH
6
NH
SH
pOH
OH
Only 14
7
Only 15
8
Only 16
10
Only 17
*Similar result when position 1 and position 2 interchanged
7

To understand the electronic effect of the charges carried by chlorine present on TCT, DFT
calculations were performed. Geometry optimization using the density functional theory (DFT)
was performed in the gas phase using Gaussian09 program package, employing the B3LYP (Becke
three parameters Lee–Yang–Parr exchange correlation functional) and the 6-311G++(d,p) as basis
set. Geometry optimization was followed by frequency calculations until no negative eigen values
were obtained. The optimized geometries were used to perform natural bond orbital (NBO)
calculations to determine the atomic charges on chlorine present in the molecule (Table 3).
Table 3: Charges carried by chlorine in TCT, m-TCTs and d-TCTs.
Natural atomic charges on Cl
Compound
Third Cl
Second Cl
First Cl
0.088
0.077
0.049
0.061
0.062
0.069
0.038
0.051
0.058
0.010
0.051
0.024
0.025
0.031
TCT
1
0.088
0.088
-
-
-
-
-
-
-
-
-
-
-
-
-
0.078
2
0.049
3
0.065
4
0.068
5
0.069
6
-
-
-
-
-
-
-
-
7
8
9
10
11
12
13
TCT carried more charge on chlorine compared to m-TCTs, thereby explaining its greater
reactivity (Table 3). Among m-TCTs, the reduction of charge was less in case of 1 (from 0.088 to
0.078) compared to 2, 3, 4 and 5 (0.049 to 0.069). This observation could be explained by the
substitution of azide (electron withdrawing) in the case of 1 compared to that of other nucleophiles
(2, 3, 4 and 5) which are electron-donating. Since 1 had a high charge on chlorine, the second
substitution at 0 ºC can be explained. Unlike 1 (0.078), the lower charge carried by “Cl” in 2
8

(0.049), 3 (0.065), 4 (0.068) and 5 (0.069) allowed the reaction to be performed at rt. Among the
d-TCTs, the charges carried by third chlorine were slightly higher when azide was one of the
substituents compared to the rest. 6 (0.038) carried a smaller charge than 7 (0.051), 8 (0.058) and
10 (0.051). In the case where azide was the third substituent (9, 11, 12 and 13), the charge carried
by chlorine decreased dramatically. This observation may explain longer reaction times in these
cases. For 9 and 11, no product formation was observed as the charge on chlorine was significantly
lower, hence deactivating the ring towards the azide substitution under the given reaction
conditions.
From the first part of this study, we can conclude that the introduction of the azide in first position
is very beneficial for the two-remaining position. Thus, to expand the applicability of the TCT-N3
(1) core in a more biological context peptides were used as nucleophiles. For this purpose,
pentapeptides (Ac-X-GGFL-NH2), synthesized using SPPS, where X = Lys, Tyr or Cys, were
sequentially incorporated to the TCT-N3 (Scheme 3). Ser was not attempted due to the poor
reactivity of the alcohol as demonstrated in the first part of this study and reported previously [14,
18]. The reactivity of the pentapeptides was studied with TCT-N3 (1) using K2CO3 as base in
ACN/water mixture as solvent in contrast of the model studies where ethyl acetate was used
(Scheme 3).
9

N3
N
O
N
N
O
NH
H
O
N
O
H2N
HN
O
NH2
O
O
NH2
HN
O
N
H
HN
N
H
H
O
NH
O
NH
NH
N
O
O
N
O
H
N
H
O
HN
HN
N
H
O
O
O
TCT-N3-Lys-Lys
O
O
NH
HN
NH
O
N
O
NH
H
N
N
N
O
NH
O
NH2
N
O
N3
O
O
O
NH2
O
N3
N
H
N
H
O
N
O
N
N
N
O
NH
O
H
H
N
O
H2N
HN
O
O
N
N
HN
Cl
N
H
HN
NH2
O
N3
S
O
N3
O
N
H
H
O
NH
N
O
HN
HN
TCT-N3-Lys
O
N
O
N
N
H
NH
O
HN
O
Cl
N
O
O
O
TCT-N3-Cys-Tyr
O
NH
H
N
NH
O
O
N3
N3
H2N
O
O
HN
N
O
NH
O
N
N
N
O
N
H
TCT-N3-Tyr-Tyr
H2N
HN
O
HN
HN
O
S
Cl
N
Cl
N
HN
Cl
NH
O
HN
TCT-N3 (1)
N
N
O
O
H
O
N
S
N
O
N3
O
N
O
H
TCT-N3-Cys
HN
O
O
NH
O
NH
H2N
TCT-N3-Tyr
O
HN
N3
NH2
NH
NH
HN
N
N
O
NH
O
O
O
HN
N
O
O
O
O
H
H
O
NH
NH
N
N
HN
N
NH2
H
O
O
O
O
NH2
H
N
TCT-N3-Tyr-Lys
O
N
TCT-N3-Cys-Lys
H
O
Scheme 3. Reaction of Lys, Cys, Tyr containing pentapeptides with TCT-N3. Amino acid
structures represent L-configuration
An equimolar solution of 1 and Ac-XGGFL-NH2 at 0 ºC in the presence of excess K2CO3 (pH >
8) in ACN/water mixture as solvent was reacted for 30 mins. Completion of the reaction was
monitored by HPLC in all cases (Figure 3). The reaction mixture was extracted using ethyl acetate
(to remove unreacted 1) to afford pure TCT-N3-Lys, TCT-N3-Tyr and TCT-N3-Cys.
10

Figure 3. HPLC chromatogram of the TCT-N3-Peptide conjugate [5-95 % of CH3CN (0.1% TFA/
H2O (0.1 %TFA) over 15 mins]
Based on our earlier findings, once an amine is incorporated on triazine ring, only another amine
can undergo reaction for the replacement of the last chlorine on the triazine core [14, 18]. Hence,
aqueous solution of TCT-N3-Lys was collected and another equivalent of Ac-KGGFL-NH2 (Lys-
peptide) was added, followed by addition of a K2CO3 solution. This mixture was then stirred at rt
for 4 days to form TCT-N3-Lys-Lys. The reaction was monitored by HPLC (Figure 4).
11

Figure 4. HPLC chromatogram of TCT-N3-Lys-Lys synthesis [5-95 % of CH3CN (0.1% TFA/
H2O (0.1 %TFA) over 15 mins]
However, when the TCT-N3 is substituted with Tyr and Cys peptides, the possibilities are
broadening. TCT-N3-Tyr was reacted in parallel with Tyr-peptide and Lys-peptide in the presence
of K3CO3 using water as solvent at rt to replace the last chlorine. The reaction with Lys-peptide
was completed in 4 h and reaction with Tyr-peptide in 8 h (Figure 5). Similarly, TCT-N3-Cys was
reacted in parallel with Lys-peptide and Tyr-peptide at rt in the presence of K2CO3 using water as
solvent for 2 days until the HPLC showed complete consumption of reactants (Figure 6). These
reactions yielded TCT-N3-Cys-Lys and TCT-N3-Cys-Tyr, respectively.
12

Figure 5. HPLC chromatogram of TCT-N3-Tyr-Tyr and TCT-N3-Tyr-Lys synthesis [20-70 % of
CH3CN (0.1% TFA/ H2O (0.1 %TFA) over 15 mins]
Figure 6. HPLC chromatogram of TCT-N3-Cys-Tyr and TCT-N3-Cys-Lys synthesis [5-95 % of
CH3CN (0.1% TFA/ H2O (0.1 %TFA) over 15 mins]
13

Peptides/proteins usually hold two different amines (in terms of reactivity) (α-NH2 in amino acids
and ε-NH2 in Lys side chain). To check the reactivity of 1 against these two amino groups, another
pentapeptide (H-KGGFL-NH2) was synthesized. This peptide, which bears free both α-NH2 and
ε-NH2 functions, was reacted with 1 (3 eq.) in the presence of K2CO3 as base at 0 °C for 30 mins.
Although in principle the α-NH2 is less reactive than the ε-NH2, HPLC monitoring demonstrated
similar reactivity of 1 towards the two amines, as double incorporation was observed. Excess of 1
was easily removed by washing reaction mixture with ethyl acetate, thus affording the product in
aqueous layer (Figure 7).
O
O
O
H
H
H
O
O
O
N3
N
Cl
N
N
N
N
H
H
N
N
NH2
H2N
NH2
N
N
H
H
N
N
NH2
N
N
N
N
O
O
H
H
+
O
O
Cl
Cl
N3
N
Cl
N
NH
TCT-N3-K(TCT-N3)GGFL-NH2
TCT-N3
H-KGGFL-NH2
N
N3
Figure 7. Reaction of 1 with α and ε-NH2 in Lys in pentapeptide (H-KGGFL-NH2) [5-95 % of
CH3CN (0.1% TFA/ H2O (0.1 %TFA) over 15 mins]
14

Conclusions
In our current efforts to develop biologically compatible linkers, azido triazine derivatives emerge
as useful tool for bioconjugation. Thus, we have demonstrated that the first chlorine in azido-
dichloro-s-triazine (1) can be easily replaced with an amino, phenol or thiol compound at 0 °C and
the second one at rt. However, if amine is introduced first, only a second amine can be incorporated
at rt as no other nucleophiles can be accommodated. If phenol or thiol is introduced in 1, amine
can easily replace the last chlorine at rt, a condition compatible with biological systems. In
addition, amine as nucleophile is highly selective upon reaction with 1 in the presence of other
nucleophiles like phenol and thiol.
We exploited this selectivity further for the reaction of pentapeptides (Ac-XGGFL-NH2 where X
= Lys or Tyr or Cys) with 1 in the presence of K2CO3 as models of other biological systems. We
demonstrate the incorporation of Lys- or Tyr- or Cys-bearing pentapeptide onto 1 at 0 °C and then
Tyr- and Lys-peptide at rt using aqueous solvents under mild conditions. The reactivity of 1 was
similar towards the α and ε amine group present in the peptide. Based on our results, which support
the applicability of azido-dichloro-triazine (1), we propose that this molecule can be further
exploited as a linker in bioconjugation involving peptides/proteins
Experimental
General
All the chemicals and diisopropylethylamine were purchased from Sigma-Aldrich (Sigma-
Aldrich, Germany). Fmoc-L-amino acids (from IRIS, Germany) were used for Solid Phase Peptide
Synthesis (SPPS). The solvents used were of analytical and HPLC reagent grade. Magnetic
resonance spectra (1H and 13C) were recorded with Bruker 400 MHz spectrometer (Bruker,
Billerica, MA, USA), and chemical shift values are reported in δ units (ppm) using TMS as internal
standard. Follow-up of the reactions and checks of the purity of the compounds were done by TLC
on silica-gel-protected aluminum sheets 60 F254 (Merck, Germany), and the spots were detected
by exposure to UV light at λ = 254 nm. Analytical HPLC was performed on an Agilent 1100
system using a Phenomenax C18 column (3μm, 4.6×50 mm) by dissolving the sample in CH3CN
only. Chemstation software was used for data processing. Buffer A: 0.1% TFA in H2O, buffer B:
0.1% TFA in CH3CN were used in HPLC. High resolution mass spectrometry (HRMS) was
15

performed using a Bruker ESI-QTOF mass spectrometer (Bruker, Billerica, MA, USA) in positive-
ion mode.
Synthetic protocol
Synthesis of 2-azido-4,6-dichloro-1,3,5-triazine (1)
The procedure for the synthesis of TCT-N3 was followed as explained in our earlier work [18].
Synthesis of mono nucleophilic substituted derivatives
TCT (0.27 mmol) was dissolved in EtOAc (1 mL) and cooled to 0 ⁰C for 5 min. Nucleophile (0.27
mmol) was then added to the above stirring solution, followed by addition of DIEA (0.27 mmol).
The reaction was stirred at 0 ⁰C (-20 ⁰C in the case of phenol) for 30 min. The progress of the
reaction was monitored by TLC (EtOAc/hexane (1:4) as mobile phase), until no starting material
was observed. The solution was washed several times with water to remove DIEA salts. The
organic layer was collected, dried over MgSO4, filtered and concentrated to afford pure product,
which was used for the next step without further purification. Compounds 3, 4 and 5 have been
reported in our earlier publication [18].
4,6-dichloro-N-isopentyl-1,3,5-triazin-2-amine (2)
Yield = 61 mg (96 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 7.7 min; 1H NMR (400 MHz, CDCl3): 0.88 (d, J = 6.6 Hz, -CH3, 6H),
1.40 (m, -CH2, 2H), 1.59 (m, -CH, 1H), 3.42 (m, -CH2, 1H), 5.70 (brs, -NH, 1H); 13C NMR (100
MHz, CDCl3): 22.4, 25.7, 37.9, 39.9, 165.8, 171.0; LCMS m/z: calcd. for C8H12Cl2N4: [M+H]+
235.05, [M+CH3CN+H]+ 278.07; found: 235.12 [M+H]+, 278.16 [M+CH3CN+H]+.
Second substitution in the case of mono-azide TCT derivative
TCT-N3 (0.27 mmol) was dissolved in EtOAc (1 mL) and cooled to 0 ⁰C for 5 min. Nucleophile
(0.27 mmol) was then added to the above stirring solution, followed by addition of DIEA (0.27
mmol). The reaction was stirred at 0 ⁰C (-20 ⁰C in the case of phenol) for 30 min. The progress of
the reaction was monitored by TLC (EtOAc/hexane (1:4) as mobile phase) until no starting
material was observed. The solution was washed several times with water to remove DIEA salts.
The organic layer was collected, dried over MgSO4, filtered and concentrated to afford pure
16

product, which was used for the next step without further purification. Compounds 7 and 8 have
been reported in our earlier publication [18].
4-azido-6-chloro-N-isopentyl-1,3,5-triazin-2-amine (6)
Yield = 61 mg (94 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 8.2 min; 1H NMR (400 MHz, CDCl3): 0.94 (d, J = 6.6 Hz, -CH3, 6H),
1.50 (m, -CH2, 2H), 1.64 (m, -CH, 1H), 3.49 (m, -CH2, 2H), 6.59 (brs, -NH, 1H); 13C NMR (100
MHz, CDCl3): 22.4, 25.7, 37.9, 39.6, 166.2, 170.2. HRMS: m/z: calcd. for C8H12ClN7: [M+H]+
242.0915; found: 242.0926.
Second substitution in the case of m-TCT derivatives
Isopentyl amine (0.27 mmol) was added to m-TCT (0.27 mmol) in EtOAc (1 mL), followed by
addition of DIEA (0.27 mmol). The reaction was stirred at room temperature for 3 h (30 mins at 0
⁰C in the case of azide and overnight for alcohol). The progress of the reaction was monitored by
TLC (EtOAc/hexane (1:4) as mobile phase) until no starting material was observed. The solution
was concentrated to dryness and the residue was dissolved in DCM and washed several times with
water to remove DIEA salts. The organic layer was collected, dried over MgSO4, filtered and
concentrated to afford pure product, which was used for the next step without further purification.
10 and 12 have been reported in our earlier publication [17, 18].
6-chloro-N2,N4-diisopentyl-1,3,5-triazine-2,4-diamine (9)
Yield = 69 mg (89 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 10.4 min; 1H NMR (400 MHz, CDCl3): 0.95 (d, J = 6.6 Hz, -CH3, 12H),
1.49 (m, -CH2, 4H), 1.66 (m, -CH, 2H), 3.49 (q, J = 8.2 Hz, -CH2, 4H), 5.79 (brs, -NH, 2H); 13C
NMR (100 MHz, CDCl3): 22.2, 24.6, 36.8, 69.0, 170.9, 172.3. HRMS: m/z: calcd. for C13H24ClN5:
[M+H]+ 286.1799; found: 286.1815.
4-chloro-N-isopentyl-6-(isopentyloxy)-1,3,5-triazin-2-amine (11)
Yield = 67 mg (87 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 10.5 min; 1H NMR (400 MHz, CDCl3): 0.92 (d, J = 6.5 Hz, -CH3, 12H),
1.44 (m, -CH2, 2H), 1.64 (m, -CH, 1H), 1.71 (m, -CH, 2H), 1.80 (m, -CH2, 2H), 3.41 (m, -CH2,
2H), 4.28 (m, -CH2, 1H), 5.08 (m, -CH2, 1H); 13C NMR (100 MHz, CDCl3): 22.4, 22.5, 24.8, 25.7,
17

37.7, 38.7, 39.0, 65.1, 166.9, 167.3, 188.0. HRMS: m/z: calcd. for C13H23ClN4: [M+H]+ 287.1653;
found: 287.1667.
4-chloro-N-isopentyl-6-phenoxy-1,3,5-triazin-2-amine (13)
Yield = 73 mg (93 %); Off-white semi-solid; 1H NMR (400 MHz, CDCl3): 0.91 (d, J = 6.5 Hz, -
CH3, 6H), 1.63 (m, -CH2, 2H), 1.69 (m, -CH, 1H), 4.43 (m, -CH2, 2H), 7.17 (m, Ar-H, 2H), 7.28
(m, Ar-H, 1H), 7.43 (m, Ar-H, 2H); 13C NMR (100 MHz, CDCl3): 22.4, 24.7, 30.9, 36.9, 121.3,
126.4, 129.7, 151.5, 172.2, 172.4, 173.2. HRMS: m/z: calcd. for C14H17ClN4O: [M+H]+ 293.1163;
found: 293.1176.
Tri-substitution in the case of d-TCT derivatives
Nucleophile (0.26 mmol) was added to a stirring solution of d-TCT (0.26 mmol) in EtOAc (1 mL),
followed by addition of DIEA (0.26 mmol). The reaction was stirred at room temperature (40 ⁰C
in the case of alcohol as substituent) for 16 h. The progress of the reaction was monitored by TLC
(EtOAc/hexane (1:5) as mobile phase) until no starting material was observed. The solution was
washed several times with water to remove DIEA salts. The organic layer was collected, dried
over MgSO4, filtered and concentrated to afford pure product.
6-azido-N2,N4-diisopentyl-1,3,5-triazine-2,4-diamine (14)
Yield = 72 mg (91 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 8.2 min; 1H NMR (400 MHz, CDCl3): 0.93 (d, J = 6.4 Hz, -CH3, 12H),
1.45 (m, -CH2, 4H), 1.66 (m, -CH, 2H), 3.41 (m, -CH2, 4H), 5.19 (brs, -NH, 1H), 5.19 (brs, -NH,
13
1H); C NMR (100 MHz, CDCl3): 22.5, 25.7, 29.7, 39.1, 166.5, 168.1. HRMS: m/z: calcd. for
C13H24N8: [M+H]+ 293.2197; found: 293.2206.
4-azido-N-isopentyl-6-(isopentylthio)-1,3,5-triazin-2-amine (15)
Yield = 78 mg (94 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 12.4 min; 1H NMR (400 MHz, CDCl3): 0.94 (d, J = 6.5 Hz, -CH3, 12H),
1.47 (m, -CH2, 2H), 1.58 (m, -CH, 1H), 1.65 (m, -CH2, 2H), 1.73 (m, -CH, 1H), 3.09 (m, -CH2,
2H), 3.45 (m, -CH2, 2H), 5.60 (brs, -NH, 1H); 13C NMR (100 MHz, CDCl3): 22.3, 25.7, 27.6, 27.7,
28.2, 28.4, 38.3, 39.4, 164.8, 168.2, 183.0. HRMS: m/z: calcd. for C13H23N7S: [M+H]+ 310.1828;
found: 310.1836.
4-azido-N-isopentyl-6-phenoxy-1,3,5-triazin-2-amine (16)
18

Yield = 73 mg (91 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 8.9 min; 1H NMR (400 MHz, CDCl3): 0.92 (d, J = 6.5 Hz, -CH3, 6H),
1.43 (m, -CH2, 2H), 1.63 (m, -CH, 1H), 3.41 (m, -CH2, 2H), 5.96 (brs, -NH, 1H), 7.16 (m, Ar-H,
2H), 7.25 (m, Ar-H, 1H), 7.39 (m, Ar-H, 2H); 13C NMR (100 MHz, CDCl3): 22.4, 25.6, 38.1, 39.4,
121.8, 125.7, 129.4, 151.9, 167.7, 170.4, 171.7. HRMS: m/z: calcd. for C14H17N7O: [M+H]+
300.1567; found: 300.1588.
4-azido-N-isopentyl-6-(isopentyloxy)-1,3,5-triazin-2-amine (17)
Yield = 71 mg (90 %); Off-white semi-solid; HPLC [30-95 % of CH3CN (0.1% TFA/ H2O (0.1
%TFA) over 15 mins] tR = 10.6 min; 1H NMR (400 MHz, CDCl3): 0.94 (d, J = 6.3 Hz, -CH3, 12H),
1.48 (m, -CH2, 2H), 1.62 (m, -CH, 1H), 1.68 (m, -CH2, 2H), 1.81 (m, -CH, 1H), 3.48 (m, -CH2,
2H), 4.33 (m, -CH2, 1H), 4.41 (m, -CH2, 1H), 6.00 (brs, -NH, 1H); 13C NMR (100 MHz, CDCl3):
22.4, 22.5, 24.8, 25.7, 37.4, 38.2, 39.3, 66.4, 167.6, 169.8, 171.6. HRMS: m/z: calcd. for
C13H23N7O: [M+H]+ 294.2053; found: 294.2069.
Synthesis of pentapeptide (Ac-XGGFL-NH2 and H-KGGFL-NH2)
SPPS was carried out manually in plastic syringe fitted with polyethylene porous using Fmoc/tBu
methodology. Fmoc-Rink-Amide AM resin (0.74 mmol/g) was washed with DMF (2 × 5 min).
Deprotection of the Fmoc group was achieved by treatment of the resin with 20% piperidine/DMF
(1 × 1 min and 1 × 7 min) followed by washing with DMF. The protected Fmoc-amino acids (3
eq.) were incorporated using DIC (3 eq.) and OxymaPure (3 eq.) in DMF, as a coupling system,
30 mins at rt. This was repeated until the tetrapeptide (H-GGFL-NH2) was achieved. Washes
between couplings and deprotections were performed with DMF (3 × 1 min). The tetrapeptidyl
resin was divided into four parts of which two parts were coupled with Fmoc-Lys(Boc)-OH, one
part coupled with Fmoc-Cys(Trt)-OH and last one with Fmoc-Tyr(tBu)-OH using above coupling
condition. Fmoc was removed in all the four cases followed by washing. Pentapeptides (H-
KGGFL-NH2, H-CGGFL-NH2 and H-YGGFL-NH2) were acetylated (acetic anhydride and DIEA
in 1:2 ratio using DMF as solvent) for 30 mins. All the peptides were dried and cleaved from resin
by treating with TFA/TIS/H2O (95:2.5:2.5) for 1 h at rt. The cleavage mixture was evaporated with
a stream of nitrogen, precipitated with Et2O, centrifuged to afford desired peptide as confirmed by
HPLC and LCMS (Table 4).
19

Synthesis of mono-substituted TCT-N3 with Ac-XGGFL-NH2
A stock solution of TCT-N3 (10 mg/mL in ACN), Ac-XGGFL-NH2 (5 mg/mL in water) and 100
mM aqueous K2CO3 solution was prepared. 1.1 eq of TCT-N3 was cooled to 0 °C. To the stirring
solution of TCT-N3 was added 1 eq of peptide solution followed by 5 eq of K2CO3 solution (pH >
8). The reaction was stirred at 0 °C for 30 mins. The progress of the reaction was monitored by
HPLC. After the completion of reaction, reaction mixture was washed with ethyl acetate to remove
excess unreacted TCT-N3 to afford TCT-N3-Lys, TCT-N3-Tyr and TCT-N3-Cys in aqueous layer.
The products were confirmed by LCMS (Table 4).
Synthesis of di-substituted TCT-N3 with Ac-XGGFL-NH2
To the mono-substituted TCT-N3 (1 eq each of TCT-N3-Lys, TCT-N3-Tyr and TCT-N3-Cys) was
added 1 eq of Ac-XGGFL-NH2 with 5 eq of K2CO3. The reaction was stirred at rt. The progress
of reaction was monitored by HPLC. The reaction time was found to vary in each case. The
reaction for synthesis of TCT-N3-Lys-Lys was found to complete in 4 days. TCT-N3-Tyr-Tyr and
TCT-N3-Tyr-Lys was found to be complete in 8 h and 4 h, respectively. TCT-N3-Cys-Tyr and
TCT-N3-Cys-Lys were formed in 2 days. All the di-substituted TCT-N3 were confirmed by LCMS
(Table 4).
Reaction of TCT-N3 and H-KGGFL-NH2
3 eq of TCT-N3 was cooled to 0 °C. To the stirring solution of TCT-N3 was added 1 eq of H-
KGGFL-NH2 solution followed by 5 eq of K2CO3 solution (pH > 8). The reaction was stirred at 0
°C for 30 mins. The progress of the reaction was monitored by HPLC. After the completion of
reaction, reaction mixture was washed with ethyl acetate to remove excess unreacted TCT-N3 to
afford TCT-N3-K(TCT-N3)-GGFL-NH2 in aqueous layer. The product was confirmed by LCMS
(Table 4).
Table 4. HPLC (5-95% B into A in 15 mins) and LCMS for peptide conjugates with TCT-N3.
Sl.
No
HPLC
(tR in min)
Expected Mass
M+[H]+
%
Compound
Found Mass
Conversion
1
2
3
Ac-KGGFL-NH2
Ac-YGGFL-NH2
Ac-CGGFL-NH2
5.6
6.9
6.5
562.69
597.69
537.65
562.37#, 282.01##
597.35#
-
-
-
537.24#
20

4
5
6
7
8
9
H-KGGFL-NH2
TCT-N3-Lys
5.0
8.2
520.65
716.20
751.20
691.17
1241.43
1276.43
1311.43
1216.39
1251.39
828.68
520.39#, 260.95##
716.21#, 358.96##
751.28#
-
100
100
100
100
100
100
100
100
100
TCT-N3-Tyr
9.1*
8.1
TCT-N3-Cys
691.24#
TCT-N3-Lys-Lys
TCT-N3-Tyr-Lys
8.1
621.28##, 427.68###
639.09##
8.3*
8.9*
8.1
10 TCT-N3-Tyr-Tyr
11 TCT-N3-Cys-Lys
12 TCT-N3-Cys-Tyr
656.78##
609.07##
8.5
626.31##
TCT-N3-K(TCT-
13
9.6
828.36#, 414.73##
N3)-GGFL-NH2
*20-70% of B into A for 15 mins; #M+[H]+, ##[M+2H]+/2, ###[M+3H]+/3
Acknowledgements
The work was funded in part by the National Research Foundation (NRF) (# 105892 and Blue Sky’s
Research Programme # 120386) The authors extend their appreciation to the Researchers Supporting
Project at King Saud University, Riyadh, Saudi Arabia for funding this work through Project No. RSP-
2020/50.
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23

Mark D. Distefano
Editor-in-Chief
Bioorganic Chemistry
University of Minnesota, Minneapolis 55455,
Minnesota, United States
August 3, 2020
Dear Professor Distefano,
It is a pleasure for us to send you the manuscript entitled “Exploiting Azido-diChloro-Triazine as
a Linker for Regioselective Incorporation of Peptides through their N, O, S functional groups” by
Anamika Sharma, Ashish Kumar, Ayman El-Faham, Beatriz G. de la Torre, and Fernando Albericio
for your consideration for publication as a full paper in Bioorganic Chemistry.
HIGHLIGHTS







1,3,5-Trichlorotriazine (TCT) is a tridentate linker that can accommodate three distinct
nucleophiles.
Azido triazine facilitates the incorporation of different nucleophiles (amine, thiol and
phenol).
The replacement of first chlorine was performed at 0 °C while that of the last chlorine
was achieved successfully at rt.
The proof of concept of this strategy for biological studies has been carried out by
incorporation of pentapeptides (Ac-XGGFL-NH2 where X = Lys or Tyr or Cys)
The azido-dichloro triazine has replaced the first and second chlorine at 0 °C and at rt,
respectively.
The reactivity of azido-dichloro triazine was found to be similar for the α and ε amine
group present in same peptide.
These findings demonstrate the applicability of azido-dichloro triazine as a linker with
potential further application in bioconjugation.
24

Exploiting Azido-diChloro-Triazine as a Linker for Regioselective Incorporation of
Peptides through their N, O, S functional groups
N
= Lys , Tyr, Cys
Ac - P e p t i d e s
=
N
N3
N
25