Unveiling and tackling guanidinium peptide coupling reagent side reactions towards the development of peptide-drug conjugates

Article abstract of DOI:10.1039/c7ra06655d

Peptide coupling reagents and especially uronium/guanidinium salts have been extensively utilized in solid-phase peptide synthesis. However, the impact of these reagents in solution phase synthesis, normally used in the formation of peptide-drug conjugates (PDCs), has not been fully explored. Herein, we identified that when guanidinium salts are used in classical peptide coupling conditions, besides leading to the formation of amide bonds, a uronium derivative can also be installed on specific amino acid scaffolds. The formation of this side product depends on the reaction conditions, as also on the nucleophilicity of the susceptible groups. Conditions to avoid this side product formation and a putative reaction mechanism describing its formation are reported.

Full text of DOI:10.1039/c7ra06655d

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Unveiling and tackling guanidinium peptide
coupling reagent side reactions towards the
Cite this: RSC Adv., 2017, 7, 50519
development of peptide-drug conjugates†
Eirinaios I. Vrettos,
Nisar Sayyad, Eftychia M. Mavrogiannaki, Evgenios Stylos,
Androniki D. Kostagianni, Serafim Papas, Thomas Mavromoustakos,
*
Vassiliki Theodorou and Andreas G. Tzakos
Peptide coupling reagents and especially uronium/guanidinium salts have been extensively utilized in solid-
phase peptide synthesis. However, the impact of these reagents in solution phase synthesis, normally used
in the formation of peptide-drug conjugates (PDCs), has not been fully explored. Herein, we identified that
when guanidinium salts are used in classical peptide coupling conditions, besides leading to the formation
of amide bonds, a uronium derivative can also be installed on specific amino acid scaffolds. The formation of
this side product depends on the reaction conditions, as also on the nucleophilicity of the susceptible
groups. Conditions to avoid this side product formation and a putative reaction mechanism describing its
formation are reported.
Received 14th June 2017
Accepted 16th October 2017
DOI: 10.1039/c7ra06655d
rsc.li/rsc-advances
peptide nucleic acid (PNA) analogues, HATU was successfully
used as a coupling reagent along with collidine, both in solution
and solid phase.14 Furthermore, the synthetic route towards
Introduction
An array of coupling reagents has been developed for peptide
synthesis, such as phosphonium (BOP, PyBOP etc.), carbodii-
mide (DCC) and guanidinium (HATU, HBTU etc.). Among the
most classically utilized guanidinium reagents are HATU
(N-[(dimethylamino)-1H-1,2,3-triazo[4,5-b]pyridin-1-ylmethy-
lene]-N-methylmethanaminium hexauorophosphate-N-oxide)
(1), and HBTU (N-[(1H-benzotriazol-1-yl)(dimethylamino)-
methylene]-N-methylmethanaminium hexauorophosphate-N-
oxide) (2).1–6
dehydrodidemnin B was improved by the use of HATU.15
Besides these, it has been reported16 that during a peptide
coupling reaction, in the presence of uronium coupling
reagents, a guanidino side product on the N-terminal amine
group was formed. More specically, it has been noted that in
a peptide coupling reaction, aminium salts can react with the N-
terminal amino moiety, leading to a guanidino side product
responsible for the termination of peptide chain elongation.17,18
Guanidinium coupling reagents, like HBTU and TBTU, have
been utilized to produce amine-modied analogues such as 4-
aminoquinoline, which have been evaluated for their antima-
larial potency,19 or peptide antagonists for the chemokine
receptor CXCR4.20 Similar isothiouronium pyrimidine–curcu-
min compounds have been synthesized using tetramethylth-
iourea for Golgi localization.21
Based on their structure, HBTU and HATU were originally
classied as O-uronium salts; later were revised to N-guanidi-
nium salts (Fig. 1).7
HATU8 since its innovation, has been established as
a peptide coupling reagent with enhanced reactivity, devoid of
side product(s) formation and racemization in the formed
products compared to other commonly used coupling reagents.
HATU has been applied in the macrocyclization of complicated
molecules9–11 as well as in the total synthesis of the antibiotic
himastatin,12,13 a symmetric cyclohexadepsipeptide dimer.
Furthermore, cyclodimerization or epimerization during
cyclization of all-L-tetrapeptides or all-L-pentapeptides,10 could
be surmounted by the utilization of HATU, which was found to
be more effective than TBTU or BOP. During the synthesis of
Peptide-drug conjugates (PDCs) represent
a
rapidly
emerging class of therapeutics capable to enhance the treat-
ment efficacy of medicinal compounds.22–24 In this frame, we
Section of Organic Chemistry and Biochemistry, Department of Chemistry, University
of Ioannina, Ioannina GR-45110, Greece. E-mail: agtzakos@gmail.com
† Electronic supplementary information (ESI) available: Spectroscopic data,
1H-NMR, 13C-NMR and MS analysis for all new compounds. See DOI:
10.1039/c7ra06655d
Fig. 1 Guanidinium salts of HATU and HBTU coupling reagents.
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evaluated the utilization of HATU/HBTU in the solution phase (1 ml ꢀ 2). The solid compound was dissolved in a cleavage
ꢁ
coupling of a PDC and specically of the Gonadotropin cocktail TFA/TIS/H2O (95/2.5/2.5, v/v) at 0 C and the resulting
Releasing Hormone (GnRH) agonist ([D-Lys]6-GnRH; Glp-His- reaction mixture was stirred at 0 ꢁC to rt for 3 h. The solvent was
Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH2) and the cytotoxic drug evaporated under reduced pressure, the residue was washed
gemcitabine (20,20-diuorodeoxycytidine, dFdC) which could be with diethyl ether and the crude compound was puried with
exploited for drug targeted delivery in cancer cells.24 Aer the RP-HPLC (Fig. S29†) to get the side product 5 and the desired
construction and HPLC purication of the relevant conjugate, PDC 6. MS (ESI+) m/z for (6) C72H97F2N21O19 calcd: 1597.72;
we observed the coexistence of a side product, bearing a mass of found: 799.8676 [M + 2H]2+, 533.5808 [M + 3H]3+ (Fig. S34†); and
the expected PDC plus 99 amu. A template peptide coupling for compound (5) C77H109F2N23O19 calcd: 1697.82; found:
reaction was conducted to monitor the exact location of this 849.71 [M + 2H]2+, 566.79 [M + 3H]3+. Compound 6 was also
side product using all amino acids of [D-Lys]6-GnRH sequence. characterized with NMR spectroscopy (Fig. S30–S32†) and its
The side product was assigned as a uronium moiety located on purity was evaluated via analytical RP-HPLC (Fig. S33†).
the phenol group of tyrosine. The same side product was also
General procedure for the synthesis of methyl esters of
determined to occur on the –OH group of tyrosine of the tumor amino acids (8–14). To a solution of an amino acid (8–14,
homing peptide HER2-BP1 (NH2-YWPSVTL-COOH),25 an hep- Table 1) in methanol (5 ml), thionyl chloride (10 ml) was added
tapeptide that is known for its activity against erbB2, sup- dropwise at 0 ꢁC and the reaction mixture was reuxed over-
pressing expression of the c-neu gene in breast cancer cells. night. Aer complete conversion of the starting materials,
Triggered by these observations we explored whether a relevant conrmed by TLC analysis, the reaction mixture was concen-
modication could be applied on other amino acids using trated under reduced pressure and the residue washed with
model dipeptides. We discovered that the sulydryl group of diethyl ether or hexane to obtain the pure methyl ester products
cysteine is also susceptible to this modication. The relevant 8–14 (NMR characterization presented in ESI†).
modication was also evident to take place in both cysteines of
Synthesis of compounds 15–21 (Table 1, ESI†). To a solution
the C1B5141–151 subdomain peptide (RCVRSVPSLCG) of protein of Fmoc-Ser(tBu)-OH (1 mmol) and HATU (1.5 mmol) in 1 ml
kinase C (PKC) g isozymes, that is known to suppress growth of DMF, DIPEA (5 mmol) was added and the resulting mixture was
human colon cancer cells.26 Finally, a possible mechanism for stirred at room temperature for 12 h. Solvent was removed
the formation of the reported uronium side product is depicted under high vacuum and the residue was washed with acetoni-
and the proper conditions to surmount its formation are also trile to afford the corresponding compounds 15–21.
reported.
Synthesis of compound 22 (ESI Fig. S38–S41†). To a solution
of [D-Lys]6-GnRH (9 mg, 7.1 mmol) and HATU (4.1 mg, 10.7 mmol)
in 1 ml DMF, DIPEA (6.3 ml, 35.9 mmol) was added and the
resulting mixture was stirred at room temperature for 12 h.
Solvent was removed under high vacuum and the residue was
Experimental
Materials and methods
puried via HPLC to afford compound 22. MS (ESI+) m/z for (22)
Fmoc amino acids, N-[(dimethylamino)-1H-1,2,3-triazo[4,5-b]
pyridin-1-ylmethylene]-N-methylmethanaminium
+
C
69H105N22O13 calcd: 1449.82; found: 725.4117 [M + 2H]2+
,
hexauor-
483.9434 [M + 3M]3+
.
ophosphate N-oxide (HATU), N-[(1H-benzotriazol-1-yl)(dime-
thylamino)methylene]-N-methylmethanaminium hexauoro-
phosphate N-oxide (HBTU) were purchased from Neosystem
Laboratoire (Strasbourg, France). Fmoc-D-lysine was purchased
from Chempep. Triisopropylsilane (TIS), N,N-diisopropylethyl-
amine (DIPEA), triuoroacetic acid (TFA) and phenol were
purchased from Sigma-Aldrich and used without further puri-
cation. Thionyl chloride was purchased from Acros Organics.
N,N-Dimethylformamide (DMF) was distillated over CaH2 and
Synthesis of compound 23 (ESI Fig. S45–S48†). To a solution
of Fmoc-HER2-BP1 (7 mg, 6 mmol) and HATU (3.7 mg, 9.6 mmol)
in 2 ml DMF, DIPEA (3.4 ml, 19.4 mmol) was added and the
resulting mixture was stirred at room temperature for 12 h.
Solvent was removed under reduced pressure and the residue
was puried via HPLC to afford compound 23. MS (ESI+) m/z for
(23) C63H81N10O13+ calcd: 1186.4; found: 1185.7 [M + H]+, 594.7
[M + 2H]2+
.
Synthesis of compounds 24, 25 (ESI Fig. S54–S60†). To
a solution of Fmoc-Cys-Tyr-NH2 (13 mg, 0.025 mmol) or Fmoc-
Ser-Tyr-NH2 (11.3 mg, 0.023 mmol) and HATU (39 mg,
0.102 mmol and 35 mg, 0.092 mmol, respectively) in 3 ml DMF,
DIPEA (13.4 ml, 0.077 mmol and 12 ml, 0.069 mmol, respectively)
was added and the resulting mixture was stirred at room
˚
stored under molecular sieves (4 A). High resolution ESI-MS
spectra were measured on a Thermo-Fischer Scientic Orbi-
trap XL and/or EVOQ™ Elite ER LC-TQ system.
Synthetic procedures
Synthesis of compounds 5 and 6 (Scheme 1). 50-O-Gemcita- temperature for 12 h. Solvent was removed under reduced
bine hemisuccinate (3) and [D-Lys]6-GnRH (4) were synthesized pressure and the residue was puried via HPLC to afford
as before.24 DIPEA (130 ml, 0.746 mmol) was added to a solution compound 24 (Fig. 5B) or 25 (Fig. 6B). MS (ESI+) m/z for (24)
of 3 (84 mg, 0.149 mmol) and HATU (85 mg, 0.223 mmol) in
C
37H49N7O5S2+ calcd: 703.9; found: 353.1 [M + 2H]2+ and
10 ml DMF under nitrogen. Aer 5 min, a solution of 4 (187 mg, MS (ESI+) m/z for (25) C32H38N5O6 calcd: 588.7; found: 589.5
+
0.149 mmol) in 3 ml DMF was added dropwise and stirred at [M + H]+.
room temperature for 12 h. The solvent was evaporated under
Synthesis of compound 26 (ESI Fig. S64–S66†). To a solution
reduced pressure and the residue was washed with acetonitrile of Fmoc-C1B5 (8 mg, 0.005 mmol) and HATU (13 mg, 0.034
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Table 1 Coupling of Fmoc-Ser(tBu)-OH with different methyl ester of amino acids using HATU, where (a) is the expected product and (b) is the
undesired product
HATU
HBTU
Products
Products
monitored
with mass
monitored
with mass
Comp. no.
Side chain (R)
Equivalents
a
b
Equivalents
a
b
Product
1.5
1.0
3
3
3
7
1.5
1.0
3
3
3
7
8
9
15
16
1.5
1.0
7
3
3
1.5
1.0
3
3
3
7
3
1.5
1.0
3
3
7
7
1.5
1.0
3
3
7
7
10
11
17
18
1.5
1.0
3
3
7
7
1.5
1.0
3
3
7
7
1.5
1.0
3
3
7
7
1.5
1.0
3
3
7
7
12
13
19
20
1.5
1.0
3
3
7
7
1.5
1.0
3
3
7
7
1.5
1.0
3
3
7
7
1.5
1.0
3
3
7
7
14
21
mmol) in 2 ml DMF, DIPEA (3 ml, 1.7 mmol) was added and the temperature (rt) for 12 h. The solvent was evaporated under
resulting mixture was stirred at room temperature for 12 h. reduced pressure, and the residue was washed with acetonitrile
Solvent was removed under reduced pressure and the residue (1 ml ꢀ2) to afford a white solid containing only compound 28.
was puried via HPLC to afford compound 26 (Fig. 7B). MS MS (ESI+) m/z for (28) C32H37N4O6+ calcd: 573.27; found: 574.00 [M
(ESI+) m/z for (26) C72H117N21O16S22+ calcd: 1596.9; found: 400.1 + H]+.
[M + 4H]4+
.
Peptide synthesis. All peptides in this work ([D-Lys]6-GnRH
Synthesis of compound 27 (ESI Fig. S67–S70†). To a solution (Glp-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH2; Fig. 3B and
of phenol (11 mg, 0.116 mmol) and HATU (88.8 mg, 0.23 mmol) in S35†), Fmoc-HER2-BP1 (Fmoc-Tyr-Trp-Pro-Ser-Val-Thr-Leu-
1.5 ml DMF, DIPEA (61 ml, 0.35 mmol) was added and the COOH; Fig. 4B and S42†), Fmoc-Cys-Tyr-NH2 (Fig. 5B and
resulting mixture was stirred at room temperature for 12 h. S49†), Fmoc-Ser-Tyr-NH2 (Fig. 6B and S49†) and Fmoc-
Solvent was removed under reduced pressure and the residue was C1B5 (Fmoc-Arg-Cys-Val-Arg-Ser-Val-Pro-Ser-Leu-Cys-Gly-NH2);
puried via HPLC to afford compound 27 (Fig. 8B). MS (ESI+) m/z Fig. 7A and S61†) were synthesized using standard Fmoc-/-tBu
for (27) C11H17N2O+ calcd: 193.3; found: 193.3 [M + H]+.
solid phase peptide synthesis (SPPS). Fmoc deprotection steps
Synthesis of compound 28 (ESI Fig. S71 and S72†). DIPEA were carried out with 20% piperidine in DMF (v/v) for 15 min.
(14.6 ml, 84.1 mmol) was added to a solution of Fmoc-Gly-OH (5 mg, Coupling reactions of Fmoc-amino acids were performed in
16.8 mmol) and HATU (9.6 mg, 25.2 mmol) in 1 ml DMF under DMF/CH2Cl2 using a molar ratio of amino acid/DIC/HOBt/resin
nitrogen. Aer 5 min, a solution of Tyr-OMe (3.3 mg, 16.8 mmol) in (3 : 3 : 3 : 1). The cleavage of the peptides from the resin was
0.5 ml of DMF was added dropwise and stirred at room carried out using either cocktail A: containing TFA/TIS/EDT/
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H2O in a percentage of 92.5/2.5/2.5/2.5 v/v (for peptides Fmoc-
Cys-Tyr-NH2 and Fmoc-C1B5) or B: containing TFA/TIS/H2O in
a percentage of 95/2.5/2.5 v/v (for peptides [D-Lys]6-GnRH, Fmoc-
HER2-BP1 and Fmoc-Ser-Tyr-NH2).
Results and discussion
In our former work, our group designed and reported24 the
development of an innovative strategy to produce a metaboli-
cally stable analogue of gemcitabine (Scheme 1), that could be
selectively delivered to prostate cancer cells (CaP). This was
achieved through the formation of a gemcitabine-GnRH PDC
(compound 6) that was based on the cell surface overexpression
of the Gonadotropin Releasing Hormone-Receptor (GnRH-R) in
cancer cells.
Fig. 2 ESI-MS spectra of compounds 5 and 6. The molecular ions of
compound 5 [M + 2H]2+ ¼ 849.71 and [M + 3H]3+ ¼ 566.79. The
molecular ions of compound 6 [M + 2H]2+ ¼ 800.55 and [M + 3H]3+
¼
During the liquid phase synthesis of compound 6 by the
reaction of Boc-protected 50-O-gemcitabine hemisuccinate (3)
and the 3-amino group of [D-Lys]6-GnRH (4), in the presence of
HATU as a coupling reagent, we realized the formation of an
unexpected side product (5) along with the desired product (6).
Evaluation of the mass spectrum (Fig. 2) of the reaction prod-
uct(s), indicated that in addition to the mass corresponding to
the desired PDC (6) (molecular ions [M + 2H]2+ ¼ 800.11 and
[M + 3H]3+ ¼ 534.11), the presence of an unexpected compound
that corresponded to the mass of the desired PDC plus 99.00
534.11.
drug and the peptide. We thus targeted to explore the amino
acid residue of the [D-Lys]6-GnRH sequence susceptible to this
side product formation, having in mind that bears two potent
nucleophiles (the 3-amino group of Lys6 and the hydroxyl group
of Tyr5). In order to assign this, we conducted a series of reac-
tions (Scheme 2) between the methyl ester of each individual
amino acid existed in the [D-Lys]6-GnRH peptide sequence
(8–14) and Fmoc-Ser(tBu)-OH (7) (Table 1, Fig. S1–S28†), using
the same reaction conditions, as in Scheme 1 and we monitored
the products with ESI-MS.
When Fmoc-Ser(tBu)-OH (7) was treated with HATU (1.5 eq.)
or HBTU (1.5 eq.) and DIPEA (5 eq.) the formation of a side
product was observed on the mass spectrum, but only when Tyr
or Lys were used in the template reactions (data shown in
Table 1). Specically, it became evident that regarding Tyr, the
formation of the side product enhanced the expected mass by M
+ 99 m/z, whereas for Lys by M + 98 m/z. These results indicate
that compound 5, the side product of compound 6, results from
the installation of an additional group to Tyr and not to Lys,
since its molecular ion has been enhanced by M + 99 m/z, and
not by M + 98 m/z. To further verify this nding and to exclude
amu (molecular ions [M + 2H]2+ ¼ 849.71 and [M + 3H]3+
¼
566.79).
We hypothesized that the second observed product could
correspond to the formation of a guanidinium/uronium side
product due to the covalent coupling of the guanidinium group
to the peptide. However, HATU-mediated guanidinylation has
been reported only for free amino groups of peptides. In
[D-Lys]6-GnRH the N-terminal amine is capped with pyrogluta-
mic acid and the 3-amino group of D-Lys6 was coupled to the
carboxy derivatized gemcitabine.
This led us to suggest that the putative guanidinium/
uronium group was installed in a different location since it
had not hindered the formation of the amide bond between the
Scheme 1 Synthesis of a gemcitabine-GnRH conjugate 6 and its
uronium side product 5. (a) HATU (1.5 eq.), DIPEA (5 eq.), DMF; (b) TFA/ Scheme 2 Coupling of Fmoc-Ser(tBu)-OH with tyrosine-O-methyl
TIS/H2O (95/2.5/2.5%).
ester: (a) HATU (1.5 eq.), DIPEA (5 eq.), DMF.
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the possibility of an ester bond formation instead of the ex-
pected amide bond, Fmoc-glycine (1 eq.) was reacted with Tyr-
OMe (1 eq.) using HATU (1.5 eq.)/DIPEA (5 eq.) in DMF, where
two products could be formed: an uronium form on tyrosine
(compound 28, Fig. S71†) or a guanidinium form on the amino
group (compound 29, Fig. S71†).
The mass spectrum of the received product (ESI Fig. S72†)
corroborates with the formation of compound 28 (m/z ¼ 573.27
[M + H]+) and not of compound 29 (m/z ¼ 572.26 [M + H]+).
Therefore, the possibility that the activated ester of Fmoc-
Ser(tBu)-OH reacted with the hydroxyl group of Tyr, to form an
ester linkage (Scheme 2), is ruled out by the MS analysis of the
resulted product. As it is shown in Scheme 2, a putative
compound could be 15c, bearing an ester linkage between
Fmoc-Ser(tBu)-OH and Tyr-OMe and N-terminal guanidinyla-
tion has an expected molecular mass of 658.34. Compound 15b
is the other alternative, with an expected molecular mass of
659.34, where the amide bond is formed between Fmoc-
Ser(tBu)-OH and Tyr-OMe and an uronium group is installed on
the phenol group of Tyr-OMe. ESI-MS analysis of the product
corroborates to the formation of 15b due to the recorded
molecular ion at m/z 660.14 (ESI Fig. S1 and S3†).
It is interesting to pinpoint that pure compound 6 bears
a white color, whereas compound 5 is a pale yellow solid (dark
yellow in solution phase) that is difficult to separate via HPLC
from its mixture with 6 (Fig. S29†). The end point of the HATU-
mediated coupling reaction is yellow-to-colourless.8 In our case,
the yellow color persisted till the end of the reaction that could
be an indication of the presence of the side product.
To further justify our ndings in more complex compounds
than template dipeptides, we reacted [D-Lys]6-GnRH (1 eq.) with
HATU (1.5 eq.) and DIPEA (5 eq.) in DMF and aer RP-HPLC
purication we received compound 22 (Fig. 3B and S38†).
The HRMS spectrum (Fig. 3A and S40†) of this compound
suggests the installation of two uronium along the [D-Lys]6-
GnRH sequence. On the basis of the information extracted from
Table 1, it can be suggested that the side product has been
installed both on the phenolic group of tyrosine and the 3-
amino group of lysine. This was further veried through
recording the 1H-NMR spectrum of compound 22 (Fig. 3B, black
color; Fig. S41†) and overlaying it with the relevant spectrum of
the [D-Lys]6-GnRH peptide (Fig. 3B, red color).
Fig. 3 Characterization of compound 22. (A) HRMS spectrum of
compound 22; (B) overlay of 1H-NMR spectra of compound 22 (black
color) and [D-Lys]6-GnRH (red color) in DMSO-d6 at 298 K.
color), which shows the absence of the phenol group at 9.2 ppm
and the appearance of one peak at 3 ppm referring to the methyl
groups of the side product with an integral of 12 (Fig. S48†).
Due to the fact that the previously used peptides didn't
contain cysteine residues in their sequences, we became inter-
ested to evaluate whether this modication is also applicable on
the –SH nucleophilic group of cysteine. Aer preparing the
It is evident that the absorption of the phenol group has
disappeared from 9.6 ppm, since it is occupied by the uronium
group and also two more distinct peaks appeared (bearing an
integral of 12) referring to the discrete chemical environment of
the four methyl groups of the guanidinium and uronium, at 2.9
and 3 ppm, respectively (Fig. S41†).
The same methodology was applied to another tumor
homing peptide widely used for peptide-drug conjugates
(HER2-BP1; NH2-YWPSVTL-COOH) to afford compound 23
(Fig. 4 and S45†). The ESI-MS spectrum (Fig. 4A and S47†) of this
compound suggests the installation of only one uronium group,
which probably occurred on the –OH group of tyrosine. This is
veried through the 1H-NMR spectrum of compound 23
(Fig. 4B, black color; Fig. S48†) overlaid with the relevant
Fig. 4 Characterization of compound 23. (A) ESI-MS spectrum of
compound 23; (B) overlay of 1H-NMR spectra of compound 23 (black
spectrum of the parent peptide (Fmoc-HER2-BP1, Fig. 4B, red color) and Fmoc-HER2-BP1 (red color) in DMSO-d6 at 298 K.
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dipeptide Fmoc-Cys-Tyr-NH2 and applying HATU (4 eq.) and
DIPEA (4 eq.) in DMF, we recorded the ESI-MS of the product
that responded to the mass of the parent compound (Fmoc-Cys-
Tyr-NH2) plus 2ꢀ 99.00 amu (Fig. 5A and S57†). This indicates
the installation of two aminium groups, one on cysteine and the
other on tyrosine. To verify the proposed formation of the side
product on both tyrosine and cysteine, we recorded 1H-NMR
spectra both of the native peptide (Fig. 5B, colored in red) and
compound 24 (Fig. 5B, colored in black; Fig. S59†). As it was
expected, the peak referring to the phenol group of tyrosine at
9.2 ppm and the sulfuryl group of cysteine at 2.2 ppm
disappeared.
This suggests that the aminium groups are installed on
tyrosine and cysteine, which was further veried from the
appearance of two more distinct peaks at 3 ppm derived from
the methyl groups of the two aminium moieties (Fig. S59†).
In addition, we also synthesized the dipeptide Fmoc-Ser-Tyr-
NH2 and post-processed it with HATU (4 eq.) and DIPEA (4 eq.)
in DMF to receive compound 25 (Fig. 6B and S60†).
We then recorded the ESI-MS of the resulted product where
we observed that the mass of the parent compound has
increased by 99.00 amu, which indicates the formation of
a single uronium side product (Fig. 6A and S58†). 1H-NMR
spectroscopy revealed that this modication is located only on
Fig. 6 Characterization of compound 25. (A) ESI-MS spectrum of
compound 25; (B) overlay of 1H-NMR spectra of compound 25 (black
color) and Fmoc-Ser-Tyr-NH2 (red color) in DMSO-d6 at 298 K.
the phenol group of Tyr (Fig. 6B, black color; Fig. S60†), as the cysteine residues. We selected C1B5 subdomain peptide of
peak referring to the phenol group of tyrosine at 9.1 ppm dis- protein kinase C (PKC) g isozymes, since it has been revealed to
appeared while the peak at 5 ppm, referring to the primary be effective in the attenuation of the growth of colon carcinoma
alcohol of serine, remained intact. Moreover, a distinct peak cells.26 C1B domain peptide of protein kinase C gamma signif-
with an integral of 12 has appeared at 3 ppm, which is derived icantly suppresses growth of human colon cancer cells in vitro
from the methyl groups of the uronium moiety (Fig. S60†).
These results clearly demonstrate that the modication can cell cycle arrest and apoptosis.26 The C1B5141–151 peptide has the
be applied not only to Tyr and Lys, but also to Cys. following sequence: RCVRSVPSLCG. It contains 2 cysteines
and in an in vivo mouse xenogra model through induction of
Our next step was to explore the possibility to observe this (C142 and C150) and according to our former observations we
HATU-mediated modication in bioactive peptides containing expected that the HATU-processed peptide, named compound
26, should adopt plus 2ꢀ 99 amu with respect to the MW of the
parent compound (peptide Fmoc-C1B5). Indeed, the ESI-MS
spectrum proved that the mass of the native peptide is 700.7
[M + 2H]2+ (Fig. 7A and S63†) and the HATU-processed peptide
is 400.1 [M + 4H]4+ (Fig. 7B and S66†).
Aerwards, with the aim to broaden the impact of our work
beyond traditional peptide chemistry, we used phenol, a simple
organic molecule widely used by synthetic chemists mostly as
starting material. We applied HATU (2 eq.) and DIPEA (3 eq.) in
DMF to acquire the relevant HATU-modied analogue. Upon
recording ESI-MS (Fig. 8A and S69†) and 1H-NMR spectrum
(Fig. 8B, black color; Fig. S70†) on the resulted product
(compound 27, Fig. S67†), we observed the disappearance of the
–OH peak at 9.3 ppm and the formation of one new peak at
3 ppm with an integral of 12 (Fig. S70†). This fully validates the
formation of the expected uronium product.
Judging from the above, it can be concluded that during
peptide-drug conjugation chemistry: (i) the side product can be
formed without interrupting the creation of the amide bond
between the drug and the peptide and (ii) the side product can
be positioned on the hydroxyl group of Tyr, on the primary
amine of Lys and on the sulydryl of Cys, which are initially
Fig. 5 Characterization of compound 24. (A) ESI-MS spectrum of
compound 24; (B) overlay of 1H-NMR spectra of compound 24 (black
color) and Fmoc-Cys-Tyr-NH2 (red color) in DMSO-d6 at 298 K.
ionized by DIPEA.
50524 | RSC Adv., 2017, 7, 50519–50526
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Scheme
3
Suggested mechanism for the uronium side product
formation.
Fig. 7 Characterization of compound 26. ESI-MS spectra of (A) native
peptide C1B5141–151 and (B) HATU-processed peptide: compound 26.
Then, the resulting anion HATU-N-oxide reacts further with
the initially formed activated ester (I) to the production of the
activated ester (II). The latter is then coupled with the primary
amino group of the next amino acid to obtain the desired
product (III). Excess of HATU in the reaction mixture reacts
further with the nucleophilic phenol group of Tyr to the unde-
sired product (IV), bearing the uronium group.
Conclusions
Peptide-drug conjugates have emerged as an efficient way to
selectively deliver spatiotemporally drugs in complex diseases
such as cancer.24 Such conjugations are frequently conducted in
the solution phase, utilizing guanidinium salts as coupling
reagents. Herein, we highlighted uncharted challenges that
could be met during amide bond formation utilizing guanidi-
nium salts in the solution phase. Upon usage of these reagents,
besides the formation of the expected amide bond, a new
aminium derivative, originated from the peptide coupling
reagent, can result as a side-product on the phenol group of
tyrosine (–OH), the primary amine of lysine (–NH2) and the
sulydryl group of cysteine (–SH). Both HATU and TBTU
investigated here were not devoid of the side product formation
upon using classical peptide coupling conditions, i.e. 1.5 equiv.
of the guanidinium coupling reagent. This observation has
been monitored in template dipeptide reactions, on the
Fig. 8 Characterization of compound 27. (A) ESI-MS spectrum of
compound 27; (B) overlay of 1H-NMR spectra of compound 27 (black
color) and phenol (red color) in DMSO-d6 at 298 K.
Moreover, during our efforts to avoid the formation of the synthesis of a peptide-drug conjugate and also on two peptides
reported side products, we conducted the reactions in Table 1, widely applied in PDCs. A plausible mechanism leading to the
using 1 eq. of HATU/HBTU, instead of the classical and estab- formation of this side product has been suggested. Based on
lished protocols using 1.5 eq. From the ESI-MS analysis of the this and seeking to identify conditions that could assist in
reaction products we discovered that by avoiding using excess of surmounting the formation of this side product, different
the coupling reagent, only the desired product was formed. reaction conditions were screened. We identied that the
Thus, it can be concluded that the side product is formed aer optimum conditions that should be followed to avoid the
the completion of the expected amide bond formation, since it formation of this side product is the usage of 1 eq. of guanidi-
is not detected with the use of equimolecular amount of the nium coupling reagent instead of the classically used 1.5 eq.
coupling reagent.
This diminishes both the formation of the side product as also
A possible mechanism for the formation of the uronium side the associated reaction costs. The current observations can nd
product is depicted in Scheme 3 (similar mechanism can be several applications in drug discovery, where orthogonal
applied for Lys- and Cys-mediated side products). In the rst coupling is carried out to produce drug-peptide/protein/
step, the deprotonated carboxylic group of the Fmoc-Ser(tBu)- antibody conjugates. We envisage that this side formation
OH attacks the electrophilic carbon of HATU to the formation could be also used for constructing novel compounds. We are
of the activated ester (I).
currently working along these lines in our lab.
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12 T. M. Kamenecka and S. J. Danishefsky, Chem.–Eur. J., 2001,
7, 41–63.
Conflicts of interest
13 T. M. Kamenecka and S. J. Danishefsky, Angew. Chem., Int.
Ed., 1998, 37, 2993–2995.
There are no conicts to declare.
14 R. Corradini, S. Sforza, A. Dossena, G. Palla, R. Rocchi,
F. Filira, F. Nastri and R. Marchelli, J. Chem. Soc., Perkin
Trans. 1, 2001, 2690–2696.
Acknowledgements
The authors would like to thank Dr A. K. Shaikh for recording
preliminary MS data. Dr N. Sayyad wishes to acknowledge the
‘Greek State Scholarships Foundation’ (IKY) for nancial
support for his PhD studies (2011–2015).
´
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50526 | RSC Adv., 2017, 7, 50519–50526
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