Modular synthetic approach to carboranyl-biomolecules conjugates

Article abstract of DOI:10.3390/molecules26072057

The development of novel, tumor-selective and boron-rich compounds as potential agents for use in boron neutron capture therapy (BNCT) represents a very important field in cancer treatment by radiation therapy. Here, we report the design and synthesis of two promising compounds that combine meta-carborane, a water-soluble monosaccharide and a linking unit, namely glycine or ethylenediamine, for facile coupling with various tumor-selective biomolecules bearing a free amino or carboxylic acid group. In this work, coupling experiments with two selected biomolecules, a coumarin derivative and folic acid, were included. The task of every component in this approach was carefully chosen: the carborane moiety supplies ten boron atoms, which is a tenfold increase in boron content compared to the L-boronophenylalanine (L-BPA) presently used in BNCT; the sugar moiety compensates for the hydrophobic character of the carborane; the linking unit, depending on the chosen biomolecule, acts as the connection between the tumor-selective component and the boron-rich moiety; and the respective tumor-selective biomolecule provides the necessary selectivity. This approach makes it possible to develop a modular and feasible strategy for the synthesis of readily obtainable boron-rich agents with optimized properties for potential applications in BNCT.

Full text of DOI:10.3390/molecules26072057

molecules
Article
Modular Synthetic Approach to
Carboranyl-Biomolecules Conjugates
Martin Kellert , Jan-Simon Jeshua Friedrichs, Nadine Anke Ullrich, Alexander Feinhals , Jonas Tepper,
Peter Lönnecke and Evamarie Hey-Hawkins *
Institute of Inorganic Chemistry, Faculty of Chemistry and Mineralogy, Leipzig University, Johannisallee 29,
04103 Leipzig, Germany; martin.kellert@uni-leipzig.de (M.K.); jf45guvu@studserv.uni-leipzig.de (J.-S.J.F.);
nadine-ullrich6@web.de (N.A.U.); alexander.feinhals@gmx.de (A.F.); jonas.tepper@web.de (J.T.);
loenneck@rz.uni-leipzig.de (P.L.)
*
Correspondence: hey@uni-leipzig.de; Tel.: +49-341-97-36151
Abstract: The development of novel, tumor-selective and boron-rich compounds as potential agents
for use in boron neutron capture therapy (BNCT) represents a very important field in cancer treatment
by radiation therapy. Here, we report the design and synthesis of two promising compounds that
combine meta-carborane, a water-soluble monosaccharide and a linking unit, namely glycine or
ethylenediamine, for facile coupling with various tumor-selective biomolecules bearing a free amino
or carboxylic acid group. In this work, coupling experiments with two selected biomolecules, a
coumarin derivative and folic acid, were included. The task of every component in this approach
was carefully chosen: the carborane moiety supplies ten boron atoms, which is a tenfold increase in
boron content compared to the L-boronophenylalanine (L-BPA) presently used in BNCT; the sugar
moiety compensates for the hydrophobic character of the carborane; the linking unit, depending
on the chosen biomolecule, acts as the connection between the tumor-selective component and the
boron-rich moiety; and the respective tumor-selective biomolecule provides the necessary selectivity.
This approach makes it possible to develop a modular and feasible strategy for the synthesis of
readily obtainable boron-rich agents with optimized properties for potential applications in BNCT.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Kellert, M.; Friedrichs, J.-S.J.;
Ullrich, N.A.; Feinhals, A.; Tepper, J.;
Lönnecke, P.; Hey-Hawkins, E.
Modular Synthetic Approach to
Carboranyl-Biomolecules Conjugates.
Molecules 2021, 26, 2057. https://
doi.org/10.3390/molecules26072057
Keywords: boron neutron cancer therapy (BNCT); modular approach; carboxylic acids and amines
Academic Editors: Andrea Trabocchi
and Elena Lenci
1
. Introduction
Since boron neutron capture therapy (BNCT) was ascertained to be a very promising
binary cancer treatment [ ], research has focused on the development of potent and
selective boron-containing drugs [ ]. The main advantage of this therapy is the generation
of highly cytotoxic particles comprising a high linear energy transfer (LET) ( particle and
Li particle). Their free mean path lengths of about 5 to 9 m [ ] roughly represent the
diameter of a human cell [ ]; therefore, these particles can only harm the surrounding
tissue within this radius. However, only the lighter isotope of boron, B (20% natural
abundance) [ ], produces high LET particles after irradiation with thermal neutrons [10].
Therefore, BNCT agents have to be enriched with B [11
is caused by irradiation with thermal neutrons [13 14] for which B exhibits a large capture
Received: 15 March 2021
Accepted: 30 March 2021
Published: 3 April 2021
1–4
5,6
α
µ
7,8
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
5
10
9
1
0
,
12]. Activation of the BNCT agents
10
,
−
24
2
cross section (3835 barn, 1 barn = 1
strategy to treat malignant tissue with tumor-selective boron-containing drugs [
as the thermal neutron beam can be focused on the affected area [21 24], thus generating
therapeutic particles only upon neutron irradiation. In this manner, normal tissue can
be spared and severe side effects, as known from pure radiotherapy or systemic effective
chemotherapy, can be reduced.
×
10
cm ) [
9
]. This renders BNCT a promising
6
,15–20],
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
–
The first boron-containing compounds used in clinical trials were L-boronophenylalanine
(L-BPA) and sodium borocaptate (BSH) [
5,6,10], but both compounds exhibit several draw-
4
.0/).
backs. For example, BSH and BPA do not exhibit optimized selectivity towards cancer
Molecules 2021, 26, 2057. https://doi.org/10.3390/molecules26072057
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2057
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cells (especially BSH), show a limited solubility in water (especially BPA [25]) and, in the
case of BSH, are not able to penetrate cells due to their anionic character. Therefore, their
application follows a tailored strategy where BSH is mostly applied for glioma treatment,
as the dianionic compound is able to cross the damaged blood–brain barrier adjoining the
malignant tissue in the human brain, and BPA is used as its fructose complex to overcome
the low water solubility [5,6,10,25]. Since May 2020, the company Stella Pharma [26] has
®
10
been allowed to market Steboronine [27] (generic name: Borofalan), which contains B-
enriched (99%) L-BPA as its D-sorbitol complex. This BNCT agent, in comparison to the
respective fructose complex, exhibits the advantage of being storable for about three years
and does not have to be freshly prepared for each use with retention of its GMP grade.
Therefore, the development of novel boron-containing tumor-selective agents for
application in BNCT is important to overcome these limitations [19,20]. For all com-
pounds, the basic requirements that must be fulfilled are: sufficient water solubility, low
inherent toxicity, high boron content and high tumor selectivity. Water solubility can be
increased by using charged compounds [28] or introducing hydrophilic moieties [29
Tumor-selectivity can be achieved by using essential biological nutrients, substrates like
boronated saccharides or amino acids [19 20 31], or even tumor-selective complex com-
pounds, like boron-containing antibodies [20 29 30 32 36]. A variety of different boron-
containing bioconjugates are known, including nucleosides [16], carbohydrates [37 38],
amino acids [39–41] and peptides [29 30 42 43]. One main prerequisite of BNCT especially
,30].
,
,
,
, , –
,
,
,
,
plays an essential role in this treatment, namely the selective accumulation of sufficient
amounts of 10B-containing compounds in cancerous tissues, so that the therapeutically
active particles destroy only the malignant cells without destroying healthy tissue. An
10
9 10
effective treatment requires boron concentrations of 10–30
atoms/cancer cell [ 10]. One approach focuses on compounds with a very large boron
content [29 30 42 44 45], another on the use of very selective BNCT agents over a longer
period, taking advantage of specific shuttle systems that facilitate accumulation of the com-
pound in the cells by internalization processes [17 29 30 33 46]. We pursued a combination
µ
g
B/g tumor, or 10 B-
7
,
,
, , ,
,
,
,
,
of both strategies by combining tumor-selective small peptides, such as highly selective
G protein-coupled receptor agonists, as biomolecules with meta-carborane derivatives to
increase the boron load [29,30,43,47,48]. However, very high carborane loading (more
than two carboranes attached to a peptide including 36 amino acids) results in loss of
solubility or aggregation in aqueous media and, therefore, decreased potency and higher
EC50 values [29,30]. Carbohydrate moieties, such as galactosyl groups, can be employed
to compensate the hydrophobic character of carborane clusters (up to eight modified
carboranes attached to the same peptide comprising 36 amino acids).
Here, we report the development of small molecules representing potential boron-
rich coupling partners for tumor-selective molecules based on a modular strategy [47
combining readily available starting materials, like meta-carborane, -D-galactopyranose
and glycine or ethylenediamine derivatives (compounds and in Scheme 1). Compounds
–49]
α
5
6
bearing a primary amine or carboxylic acid group represent potentially universal coupling
partners for biomolecules [48]; representative coupling experiments are also included here
to demonstrate the generalizability of this approach.

Molecules 2021, 26, 2057
3 of 16
Scheme 1. Synthesis of galactopyranosyl-modified meta-carborane-containing carboxylic acid (
as the target compounds for modular conjugation with tumor-selective biomolecules. ( ) 1.20 eq. tert-butyl glycinate
) 1.11 eq. N-tert-butoxycarbonyl-ethylenediamine, 1.12 eq.
) 1.20 eq. 1-(trifluoromethanesulfonylmethyl)-1,7-dicarba-closo-
5) and primary amine (6)
a
◦
hydrochloride, 2.96 eq. DIPEA, MeCN, 72 h, 45 C, 68%; (
b
◦
◦
DIPEA, MeCN, 15 min, 0 C, then 48 h, 40 C, 77%; (
c
◦
dodeca-borane(12), 1.20 eq. K CO , toluene, 43 h, 95 C, 54%; (d) 1.20 eq. 1-(trifluoromethanesulfonylmethyl)-1,7-dicarba-
closo-dodecaborane(12), 1.20 eq. K CO , toluene, 48 h, 98 C, 51%; (e) 55.0 eq. TFA, 3 h, rt, 65%; (f) 48.2 eq. TFA, 3 h,
2
3
◦
2
3
rt, quant.

Molecules 2021, 26, 2057
4 of 16
In this regard, the synthesis of bifunctional anticancer drugs is of special interest.
Several examples are known where drugs are used as theranostic compounds [50
,51] or
exhibit dual effects [52 53]. For example, derivatives of 7-amino-4-methylcoumarin are
,
known for their anticancer activity [54]. Thus, combination with a carborane derivative
can lead to drugs that possess anti-cancer properties and the ability to capture thermal
neutrons for applications in BNCT. Furthermore, folic acid and its derivatives, which are
already used as tumor-selective synthons for applications in BNCT [15,32,55], can act as
diagnostic probes for some solid cancer types when combined with imaging agents [56].
Thus, conjugates of folic acid with carborane derivatives and an imaging agent using both
carboxylic groups of folic acid could be used to generate highly selective BNCT agents
with excellent imaging properties.
2. Results and Discussion
The starting materials 1-(trifluoromethanesulfonylmethyl)-1,7-dicarba-closo-dodeca
borane(12) and 1,2:3,4-di-O-isopropylidene-6-deoxy-α-D-galactopyranosyl-6-triflate were
prepared according to protocols from the literature. The synthetic procedures of Goto and
co-workers for the synthesis of 1-(hydroxymethyl)-1,7-dicarba-closo-dodecaborane(12) [57
and those of Kalinin and co-workers for the preparation of 1-(trifluoromethanesulfonylmeth
yl)-1,7-dicarba-closo-dodecaborane(12) [58] were employed. 1,2:3,4-Di-O-isopropylidene-
]
α
-
D-galactopyranose is commercially available or can be prepared in quantitative yield accord-
ing to a procedure described by Saltan and co-workers [59]. 1,2:3,4-Di-O-isopropylidene-
α
-D-galactopyranose was converted to the triflate 1,2:3,4-di-O-isopropylidene-6-deoxy-
α
-D-galactopyranosyl-6-triflate following a procedure described by Brackhagen and co-
workers [60].
The next step was the introduction of the respective linking units (glycine or ethylene-
diamine) starting from the commercially available protected derivatives, namely tert-butyl
glycinate hydrochloride and N-tert-butoxycarbonyl-ethylenediamine, to prevent undesired
side reactions during the synthetic steps. The reaction with the galactopyranosyl moiety
was conducted under basic conditions using N,N-diisopropylethylamine (DIPEA) as a base
in acetonitrile (MeCN) at elevated temperature (Scheme 1a,b). Both reactions gave the
desired products, tert-butyl-N-(1,2:3,4-di-O-isopropyliden-6-deoxy-
α
-D-galactopyranos-6-
yl)glycinate (
1
) and tert-butyl-{2-[(1,2:3,4-di-O-isopropylidene-6-deoxy- -D-galactopyranos-
α
6-yl)amino]ethyl}carbamate (2) in good to excellent yields.
Beside the desired product
2
, the disubstituted compound tert-butyl-(2-{[bis(1,2:3,4-
0
di-O-isopropylidene-6-deoxy-
α
-D-galactopyranos-6-yl)]amino}ethyl)-carbamate (
2
) was
isolated with an 8% yield (see the Supplementary Materials). This product was formed
due to the increased nucleophilicity of the secondary amine in in comparison to a
2
primary amine in the starting material; the amount corresponded to the small excess of the
ethylenediamine derivative employed here.
The carboranyl moiety was introduced by reacting 1 and 2 with 1-(trifluoromethanesulf
onylmethyl)-1,7-dicarba-closo-dodecaborane(12) under basic conditions (potassium carbon-
ate) in toluene (Scheme 1c,d); for optimized reaction conditions see the Supplementary Ma-
terials. The products, tert-butyl-N-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl]-N-(1,2:3,4-
di-O-isopropylidene-6-deoxy-
dicarba-closo-dodecaboran-1-ylmethyl)-(1,2:3,4-di-O-isopropyliden-6-deoxy-
ranos-6-yl)amino]ethyl}carbamat ( ) were obtained in moderate yields (54 and 51%, respec-
α
-D-galacatopyranos-6-yl)glycinate (
3
) and tert-butyl-{2-[(1,7-
α
-D-galactopy
4
tively), presumably due to steric hindrance (bulky carboranyl moiety and secondary amine
derivative).
t
The final step was the deprotection of the acid-labile protecting groups (R(CO)O Bu
t
and RNH(CO)O Bu, where R is the organic moiety) of compounds
3 and 4 with trifluo-
roacetic acid (TFA), with formation of the volatile reaction products isobutene and carbon
dioxide leading to very pure products N-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl]-
1
N-(1,2:3,4-di-O-isopropylidene-6-deoxy-
α
-D-galacatopyranos-6-yl)glycine (
5
) and N -(1,7-
1
dicarba-closo-dodecaboran-1-yl-methyl)-N -(1,2:3,4-di-O-isopropylidene-6-deoxy-α-D-galac

Molecules 2021, 26, 2057
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topyranos-6-yl)ethane-1,2-diamine (
6
) in high yields due to a facile purification [61]. Anhy-
drous conditions must be employed to prevent cleavage of the monosaccharide protecting
groups [61]. Compound was first purified by column chromatography, which led to a
noticeable loss of product (47% yield) due to the highly polar carboxylic acid group. There-
fore, compounds and were purified by evaporation of all volatile reaction products with
5
5
6
dichloromethane (DCM) as an entrainer, leading to pure compounds in good to excellent
yields (65% and quantitative yield, respectively; details are given in the Supplementary
Materials). Apparently, deprotection of the carboxylic acid ester (tert-butyl group) is less
efficient than deprotection of the carbamate (Boc group).
The new compounds 1–6 were fully characterized by NMR spectroscopy (numbering
scheme given in Figure S1), mass spectrometry and infrared spectroscopy and showed
a purity of at least 95%. Furthermore, we were able to demonstrate that the same proce-
dure can also be applied for the synthesis of the corresponding ortho-carborane deriva-
1
1
tive N -[(1,2-dicarba-closo-dodecaboran-1-yl)methyl]-N -(1,2:3,4-di-O-isopropylidene-6-
deoxy- -D-galactopyranos-6-yl)ethane-1,2-diamine (see the Supplementary Materials for
further details).
With the glycine derivative
α
5
and ethylenediamine derivative 6 in hand, exemplary
coupling reactions with two selected biomolecules that play roles in cancer treatment
were conducted. 7-Amino-4-methylcoumarin (Figure 1, left), already employed in the
preparation of polyfunctional cancer therapeutics based on cisplatin derivatives [62] but not
for the preparation of potential BNCT agents [63
. As coupling partner for amine , folic acid (Figure 1, right) was chosen as this biomolecule
has already been employed in the synthesis of potential BNCT agents [15 32 46 55 66]. Some
–65], was selected as a coupling partner for
5
6
,
,
,
,
cancer types overexpress folate receptors on their cell membrane surfaces, which can be
used for selective uptake of the final BNCT agent in the respective cancer cells, increasing
the efficacy of the therapy [32,67].
Figure 1. 7-Amino-4-methylcoumarin (left) and folic acid (right) as two biomolecules selected to demonstrate the coupling
behavior of glycine derivative 5 and ethylenediamine derivative 6.
The strategy for coupling glycine derivative
5 with the weakly nucleophilic coumarin
derivative followed a procedure described by Quéléver and co-workers using phos-
1
phoryl chloride and pyridine [68] and resulted in N -[(1,7-dicarba-closo-dodecaborane-1-
1
2
yl)methyl]-N -(1,2:3,4-di-O-isopropylidene-6-deoxy-
α-D-galacatopyranos-6-yl)-N -[(4-meth
yl)-2-oxo-2H-chromen-7-yl)glycineamide ( ), albeit in low yield (27%) (Scheme 2). Attempts
7
to use less harsh conditions (N,N’-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccini
mide (NHS)) for this coupling reaction were not successful [69]. Using different coupling
reagents (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and hydroxybenzotri-
azol (HOBt) with N,N-diisopropylethylamine (DIPEA) as base [69]) yielded the desired
product; however, it was in a very low yield of only 18%, indicating that this method
is inferior to the phosphoryl chloride approach and the low reactivity of the coumarin
derivative is the main issue. Compound 7 was fully characterized by NMR spectroscopy,
mass spectrometry and infrared spectroscopy, proving the successful synthesis (with at
least 95% purity) of this bioconjugate as a proof of principle in this approach.

Molecules 2021, 26, 2057
6 of 16
◦
Scheme 2. (a) 1.20 eq. 7-amino-4-methylcoumarin, 1.10 eq. POCl , pyridine, 1 h, −18 C, 27%.
3
The coupling reaction between folic acid and the primary amine
6
turned out to be
more complicated due to the presence of two unprotected carboxylic acid groups in the for-
mer. Similar reactions have been reported where no additional protecting group was used
for the secondary carboxylic acid group [55
higher reactivity and, therefore, is more prone to undergo coupling reactions. However,
here, the reaction of with folic acid, following the procedure described by Trindade and co-
,66,70], as the primary COOH group exhibits a
6
workers, using DCC and NHS as activation reagents [70], gave both the desired monosub-
2
stituted species, namely N -(4-{[(2-amino-4-hydroxypteridine-6-yl)methyl]amino}benzoyl)-
5
N -{2-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl-(1,2:3,4-di-O-isopropyliden-6-deoxy-
α
-
D-galactopyranos-6-yl)amino]ethyl}-L-glutamine (8) or its isomer, and the disubstituted
species, namely (S)-2-(4-{[(2-amino-4-hydroxypteridine-6-yl)methyl]amino}benzamido)-
1
5
N ,N -bis-{2-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl-(1,2:3,4-di-O-isopropylidene-6-de
oxy- -D-galactopyranos-6-yl)amino]ethyl}amino)pentanediamide ( ) (Scheme 3), also veri-
fied by high-resolution mass spectrometry (m/z 883.5151 for and m/z 1323.8829 for ).
Obviously, in this case, the protocol from the literature [70] for conjugate formation with
folic acid was not applicable, as we got a mixture of and in an unknown ratio. However,
has a high boron contents which could be
α
9
8
9
8
9
the obtained disubstituted folic acid derivative
useful for applications as a BNCT agent.
9
Scheme 3.
(a) 1.00 eq. folic acid, 1.00 eq. DCC, 1.00 eq. NHS, 1.10 eq. DIPEA, dimethylformamide,
no yield determined.

Molecules 2021, 26, 2057
7 of 16
3. Materials and Methods
All reactions were carried out under nitrogen atmosphere using Schlenk techniques,
if not reported otherwise. Anhydrous diethyl ether and DCM were obtained with an
MBRAUN solvent purification system MB SPS-800 (M. Braun Inertgas-Systeme GmbH,
Garching, Germany). MeCN and 2,4,6-collidine were dried over calcium hydride and dis-
tilled prior to use. Anhydrous tetrahydrofuran was dried over potassium and distilled prior
to use. All solvents were stored over a molecular sieve (3 Å) under nitrogen atmosphere. 1,2-
Dicarba-closo-dodecaborane(12) and 1,7-dicarba-closo-dodecaborane(12) are commercially
available. 1-(Hydroxymethyl)-1,2-dicarba-closo-dodecaborane(12) [71], 1-(hydroxymethyl)-
1
,7-dicarba-closo-dodecaborane(12) [57], 1-(trifluoromethanesulfonylmethyl)-1,2-dicarba-
closo-dodecaborane(12) [58], 1-(trifluoromethanesulfonylmethyl)-1,7-dicarba-closo-dodecab
orane(12) [58], 1,2:3,4-di-O-isopropylidene-6-deoxy- -D-galactopyranos-6-yltriflat [60] and
-amino-4-methylcoumarin [72] were synthesized according to respective protocols from
the literature. All other chemicals were commercially available and were used as received.
α
7
Thin-layer chromatography (TLC) with silica gel 60 F2 on glass, available from
54
®
Merck KGaA (Darmstadt, Germany), or ALUGRAM XTRA SIL G/UV from Macherey-
254
Nagel GmbH & Co. KG (Düren, Germany) on aluminum foil were used for monitoring
the reactions. Carborane-containing spots were visualized with a 5% solution of PdCl2
in methanol. Non-carborane-containing spots were visualized with a basic potassium
permanganate solution. For chromatography, silica gel (60 Å) with a particle diameter in
the range of 0.035 to 0.070 mm was used. Prior to column chromatography, raw products
®
were adsorbed on Celite S from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).
NMR measurements were carried out on a Bruker AVANCE III HD spectrometer
TM
(Bruker Corporation, Billerica, MA, United States of America) with an Ascend
400 mag-
net (Bruker Corporation, Billerica, MA, United States of America) at room temperature.
1
13
1
11
Tetramethylsilane was used as internal standard for H- and C{ H}-NMR spectra; B-
and 11B{ H}-NMR spectra were referenced to the
1
Ξ
scale [73]. NMR spectra were recorded
at the following frequencies: H: 400.16 MHz, C: 100.63 MHz, B: 128.38 MHz. All chem-
ical shifts are reported in parts per million (ppm). Assignment of the H and C signals
was based on 2D-NMR spectra (H,H-COSY, H,C-HSQC, H,C-HMQC and H,C-HMBC).
NMR data were interpreted with MestReNova [74]. NMR signals that appeared as broad
1
13
11
1
13
1
11
1
11
overlapping signals with the shape of a multiplet or singlet in either H-, B{ H}- or B-
NMR spectra were described as “br” (broad). The numbering scheme of the compounds
for assignment of NMR signals is given in the Supplementary Materials (see Figure S1).
IR data were obtained with a PerkinElmer FT-IR spectrometer Spectrum 2000 (Perkin
Elmer, Inc., Waltham, MA, United States of America) as KBr pellets and with a Thermo
Scientific Nicolet iS5 with an ATR unit (Thermo Fisher Scientific, Waltham, MA, United
−
1
States of America) in the range from 4000 to 400 cm
.
High-resolution electrospray ionization mass spectrometry (ESI-HRMS) was per-
formed with an ESI ESQUIRE 3000 PLUS spectrometer (Bruker Corporation, Billerica,
MA, United States of America) with an IonTrap-analyzer from Bruker Daltonics or on a
MicroTOF spectrometer from Bruker Daltonics with a ToF analyzer in negative or positive
mode. As solvents for the measurements, DCM, MeCN, methanol or mixtures of these
solvents were used. Interpretation of the spectra was carried out using MestReNova [74].
Tert-Butyl-N-(1,2:3,4-di-O-isopropylidene-6-deoxy-α-D-galactopyranos-6-yl)glycinate (1):
Diisopropylethylamine (2.55 mL, 15.0 mmol, 2.96 eq.) was added dropwise under nitro-
gen atmosphere at room temperature to a solution of tert-butyl glycinate hydrochloride
(1.12 g, 6.12 mmol, 1.20 eq.) and 1,2:3,4-di-O-isopropylidene-6-deoxy-α-D-galactopyranos-
6
-trifluoromethanesulfonate (2.00 g, 5.10 mmol, 1.00 eq.) in 50 mL MeCN. The reaction
◦
mixture was stirred for 72 h at 45 C. The reaction was stopped by evaporating all volatile
components under reduced pressure at 45 C. The crude product was dissolved in ethyl
◦
acetate (100 mL) and washed with H O and saturated NaCl solution. Subsequently, the
2
aqueous layer was extracted with ethyl acetate (3
and the solvent was removed under reduced pressure. The crude material was puri-
×
50 mL), dried with Na SO , filtered
2 4

Molecules 2021, 26, 2057
8 of 16
fied by column chromatography using n-hexane/ethanol (14:1 (R = 0.41) to 10:1 (R =
f
f
0
.58), (v/v)) as eluent. Compound
.48 mmol). H-NMR (400 MHz, chloroform-d ):
1
was obtained as colorless oil in 68% yield (1.30 g,
1
3
δ
[ppm] = 1.33, 1.34, 1.45 and 1.54 (s,
1
0
0
0
0
2H, 1
3, 13 , 14 and 14
CH ), 1.46 (s, 9H,
10, 10 and 10 ’
CH ), 2.77 to 2.92 (m, 2H, CH ), 3.26 to
6
1
3
4
3
3
2
7
5
4
3
3
.40 (m, 2H, CH ), 3.88 (m, 1H, CH), 4.22 (dd, 1H, CH, J
.31 (dd, 1H, CH, J
4.9 Hz), 5.54 (d, 1H, CH, J
= 7.9 Hz, J
= 4.9 Hz),
2
HH
HH
2
3
3
3
3
3
= 5.1 Hz, J
= 2.4 Hz), 4.60 (dd, 1H, CH, J
= 7.9 Hz, J
HH
1
HH
HH
HH
3
13
0
1
=
= 5.1 Hz). C{ H}-NMR (100 MHz, chloroform-d ):
δ
HH
1
0
0
0
0
0
1
3,13 ,14 or 14
13,13 ,14 or 14
13,13 ,14 or 14
[ppm] = 24.5 (s,
CH ), 24.9 (s,
CH ), 26.0 (s,
CH ), 26.1
3
3
3
0
0
0
0
1
3,13 ,14 or 14
10,10 and 10 ’
4
6
7
5
(s,
CH ), 28.1 (s,
CH ), 49.1 (s, CH ), 51.8 (s, CH ), 67.1 (s, CH),
3
3
2
2
2
3
9
1
11
7
0.5 (s, CH), 70.8 (s, CH), 71.9 (s, CH), 80.9 (s, C
q
3
), 96.4 (s, CH), 108.4 (s,
CH-sp ), 2935 (m,
q
C ), 109.1 (s,
CH-sp ), 2361 (w), 1736 (s,
1
2
8
3
C
q
), 171.4 (s, C
q
). IR (KBr):

̃
= 2982 (m,
ν
ν
ν
COEster), 1459 (w, δas.CH ), 1372 (m, δs.CH ), 1256 (m), 1213 (s), 1165 (s, νas.C-O-CEster),
2 3
1
114 (m), 1070 (s,
ν
C-O-CEther), 1003 (m), 918 (w), 898 (w), 854 (w), 804 (w), 771 (w), 650
−
1
+
(w), 512 (w) cm . ESI-HRMS: (m/z) calculated for [NaC H NO ] = 396.19986; ob-
1
8
31
7
+
+
+
served 396.20153 [M+Na ] ; calculated for [C H NO ] = 374.21791; observed 374.21969
18
32
7
+
+
+
[M+H] ; calculated for [NaC H NO ] = 340.13726; observed 340.13844 [M-C H +Na] ;
14 23 7 4 8
+
+
calculated for [C H NO ] = 318.15531; observed 318.15632 [M−C H +H] .
1
4
24
7
4
8
Tert-Butyl-{2-[(1,2:3,4-di-O-isopropylidene-6-deoxy-
rbamate ( ): A 250 mL round-bottom flask was charged with 3.20 g (8.16 mmol, 0.76 eq.)
,2:3,4-di-O-isopropylidene-6-deoxy- -D-galactopyranos-6-trifluormethanesulfonate and
0 mL MeCN. Then, 1.70 mL (1.73 g, 10.8 mmol, 1.00 eq.) tert-butyl-N-(2-aminoethyl)carbamate,
α-D-galactopyranos-6-yl)amino]-ethyl}ca
2
1
6
α
dissolved in 10 mL MeCN, were added via a dropping funnel to this solution. The reaction
◦
mixture was cooled to 0 C and, subsequently, 2.05 mL (1.56 g, 12.1 mmol, 1.12 eq.) N,N-
diisopropylethylamine, dissolved in 10 mL MeCN, were added dropwise. After stirring
◦
◦
for 15 min at 0 C, the reaction mixture was warmed to 40 C and stirred for two days. The
reaction was stopped by cooling to room temperature and adding 30 mL saturated NH Cl
4
solution. All volatile components were removed under reduced pressure, and the remain-
ing aqueous layer was extracted four times with 40 mL ethyl acetate, respectively. The
combined organic layers were dried over MgSO . The drying agent was filtered off and the
4
solvent was removed under reduced pressure. The crude product was purified by column
chromatography (a: DCM/methanol, 20:1, (v/v); b: acetone; c: n-hexane/isopropanol,
8
:1 to 3:1, (v/v)). A total of 0.56 g tert-butyl-(2-{[bis(1,2:3,4-di-O-isopropylidene-6-deoxy-
α
-
0
D-galactopyranos-6-yl)]amino}ethyl)-carbamate (
2
) (R = 0.38, DCM/methanol, 20:1, (v/v),
f
0
.87 mmol, 8%, analytical data are given in the Supplementary Materials) was obtained
as a colorless solid, as well as 3.28 g tert-butyl-{2-[(1,2:3,4-di-O-isopropylidene-6-deoxy-
α
-D-galactopyranos-6-yl)amino]ethyl}carbamate (
2
) (R = 0.12 n-hexane/isopropanol, 5:1,
f
1
(
v/v), 8.27 mmol, 77%) as a colorless solid. H-NMR (400 MHz, acetone-d ):
δ
[ppm] =
6
0
0
0
0
1
3,13 ,13 ’,16,16 ,17 and 17
8,9
1.34, 1.35, 1.43 and 1.52 (s, 21H,
CH ), 3.48 to 3.61 (m, 4H, CH ), 3.58
3 2
6
5
3
3
4
3
(m, 2H, CH ), 4.28 (td, 1H, CH, J
= 6.0 Hz, J
= 1.8 Hz), 4.39 (dd, 1H, CH, J
2
HH
HH
HH
3
2
3
3
=
7.9 Hz, J
= 1.9 Hz), 4.45 (dd, 1H, CH, J
= 5.0 Hz, J
= 2.5 Hz), 4.73 (dd, 1H,
= 4.9 Hz), 6.64 (s, 1H, NH),
HH
HH
HH
3
3
3
1
3
7
CH, J
= 7.8 Hz, J
= 2.5 Hz), 5.56 (d, 1H, CH, J
HH
HH
HH
10
13
1
8
.24 (s, 1H, NH). C{ H}-NMR (100 MHz, acetone-d ):
δ
[ppm] = 24.4, 25.0, 26.2, 28.5
6
0
0
0
0
1
3, 13 , 13 ’16,16 ,17 and 17
6
8 or 9
8 or 9
(
s,
CH ), 38.2 (s, CH ), 49.3 (s,
CH ), 50.7 (s,
CH ), 64.6 (s,
3
3
2
2
2
5
CH), 71.2 (s, CH), 71.6 (s, CH), 72.2 (s, CH), 80,6 (s, 12C
2
4
1
q
), 97.1 (s, CH) 109.9 and 110.7
14, 15
11
(
s,
Cq). Carbonyl carbon atom C was not observed.
Tert-Butyl-N-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl]-N-(1,2:3,4-di-O-isopropylidene-6-
deoxy- -D-galacatopyranos-6-yl)glycinate ( ): A suspension of tert-butyl-N-(1,2:3,4-di-O-
isopropylidene-6-deoxy- -D-galactopyranos-6-yl)glycinate ( ) (0.94 g, 2.52 mmol, 1.00 eq.),
-(trifluoromethanesulfonylmethyl)-1,7-dicarba-closo-dodecaborane (0.93 g, 3.02 mmol,
α
3
α
1
1
1
.20 eq.) and potassium carbonate (0.42 g, 3.02 mmol, 1.20 eq.) in 10 mL toluene was
◦
stirred at 95 C for 43 h under nitrogen atmosphere. Then, the suspension was diluted with
ethyl acetate and washed with 20 mL H O and saturated NaCl solution. Subsequently, the
2
aqueous layer was extracted with ethyl acetate (3
×
15 mL), the combined organic layers
were dried over MgSO and filtered, and the solvent was removed under reduced pres-
4

Molecules 2021, 26, 2057
9 of 16
sure. The crude material was purified by column chromatography using n-hexane/ethyl
acetate (20:1, (v/v), R = 0.50) and then n-hexane/ethanol (20:1, (v/v), R = 0.27) as eluent.
f
f
1
Compound
3
was obtained as a colorless oil in 54% yield (721 mg, 1.36 mmol). H-NMR
400 MHz, chloroform-d ):
CH ), 1.50 to 3.00 (br m, 10H, 10 BH), 2.83 to 2.91 (m, 1H, CHH), 2.89 (s, 1H,
CH), 2.97 to 3.09 (dd, 1H, CHH, J
0
0
(
δ
[ppm] = 1.34, 1.42 and 1.56 (s, 12H, 1
3,13 ,14 and 14
CH ), 1.44 (s,
1
3
0
0
1
0,10 and 10 ’
6
9
H,
3
1
7
6
2
3
15
2
= 14.1 Hz, J
= 7.0 Hz), 3.13 (d, 1H, CHH, J
HH
HH HH
15
3
7
3
=
15.7 Hz), 3.23 (d, 1H, CHH, J
= 15.7 Hz), 3.36 (d, 1H, CHH, J
= 17.8 Hz), 3.53
= 7.9 Hz, J
HH
HH
7
3
5
4
3
3
(d, 1H, CHH, J
= 17.8 Hz), 3.84 (m, 1H, CH), 4.25 (dd, 1H, CH, J
=
HH
HH
3
HH
2
3
3
3
1
.9 Hz), 4.29 (dd, 1H, CH, J
= 5.1 Hz, J
= 2.4 Hz), 4.60 (dd, 1H, CH, J
= 7.9 Hz,
HH
HH
HH
3
1
3
13
1
JHH = 2.4 Hz), 5.50 (d, 1H, CH, J
[ppm] = 24.6, 24.9, 26.0 (s,
s, CH ), 55.9 (s, CH ), 60.9 (s, CH ), 66.8 (s, CH), 70.4 (s, CH), 70.8 (s, CH), 71.9
= 5.1 Hz). C{ H}-NMR (100 MHz, chloroform-d ):
HH
1
0
0
0
0
1
3,13 ,14 and 14
10,10 and 10 ’
17
δ
CH ), 28.2 (s,
CH ), 54.8 (s, CH), 55.0
3
3
6
7
15
5
2
3
(
(
2
2
2
4
16
9
1
11, 12
s, CH), 78.5 (s,
C
q
), 81.5 (s, C
q
), 96.5 (s, CH), 108.5 and 109.1 (s,
[ppm] = 15.7 (s, 2B),
q
C ), 170.8 (s,
13.6 (s, 2B),
8
11
1
C
q
). B{ H}-NMR (128 MHz, chloroform-d ):
δ
−
−
−
10.9
1
+
(
5
s, 4B),
−
9.5 (s, 1B),
52.3941; observed 552.3966 [M+Na] .
Tert-Butyl-{2-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl-(1,2:3,4-di-O-isopropyliden-6-deox
-D-galactopyranos-6-yl)amino]ethyl}carbamat ( ): A total of 1.66 g (4.12 mmol, 1.00 eq.) tert-
-D-galactopyranos-6-yl)amino]-ethyl}carba
) was placed in a 50 mL round-bottom flask and dissolved in 10 mL toluene. Subse-
quently, 1.51 g (4.94 mmol, 1.20 eq.) 1-(trifluoromethanesulfonylmethyl)-1,7-dicarba-closo-
−
4.4 (s, 1B). ESI-HRMS: (m/z) calculated for [NaC H B NO ] =
2
1
43 10
7
+
y-
α
4
α
butyl-{2-[(1,2:3,4-di-O-isopropylidene-6-deoxy-
mate (
2
dodecaborane(12), dissolved in 10 mL toluene, and 0.68 g (4.94 mmol, 1.20 eq.) K CO
2
3
◦
were added. The suspension was heated to 98 C and stirred for 48 h. The reaction was
stopped by adding 20 mL distilled water and 4 mL saturated NaCl solution. The aqueous
layer and organic layer were separated. The aqueous layer was extracted two times with
2
0 mL ethyl acetate. The combined organic layers were dried over MgSO , the drying
4
agent was filtered off and the solvent was removed under reduced pressure. The resulting
yellow-brownish oil was purified by column chromatography (n-hexane/ethyl acetate,
5
:1, (v/v)) and
4
(1.17 g, 2.09 mmol, 51%, R = 0.24 (n-hexane/ethyl acetate, 5:1, (v/v))) was
f
1
isolated as a colorless foamy solid. H-NMR (400 MHz, chloroform-d ):
δ
[ppm] = 1.19 to
3
1
0
0
1
5,15 ,16 and 16
3
.41 (m, br, 10H, 10 BH), 1.34, 1.36, 1.45 and 1.56 (s, 12H,
CH ), 1.45 (s, 9H,
CHH), 2.63 to 2.72 (m, 1H, CHH), 2.80 to 2.88
0
0
1
2, 12 and 12 ’
7 or 8
6
CH ), 2.55 to 2.63 (m, 1H,
3
m, 2H, 7 or 8CHH and CHH), 2.90 (s, 1H, CH), 2.94 to 3.07 (m, 2H, CHH and CHH),
6
19
17
17
(
7
and 8
5
4
3
3
.10 to 3.25 (m, 2H,
CHH), 3.85 (m, 1H, CH), 4.24 (d, 1H, CH, J
= 7.9 Hz), 4.31
HH
2
3
3
3
3
3
(dd, 1H, CH, J
= 5.1 Hz, J
= 2.4 Hz), 4.62 (dd, 1H, CH, J
= 7.9 Hz, J
=
HH
HH
HH
HH
9
1
3
13
1
2
.4 Hz), 5.35 (s, br, 1H, NH), 5.51 (d, 1H, CH, J
= 5.1 Hz). C{ H}-NMR (100 MHz,
HH
0
0
0
0
1
5,15 ,16 and 16
12,12 and 12 ’
17
chloroform-d ):
δ
[ppm] = 24.4, 24.9, 26.0 (s,
CH ), 28.5 (s,
CH3),
1
3
8.4 (s, 7 CH ), 53.0 (s, CH ), 54.6 (s,
or 8
6
7 or 8
4
CH ) 54.7 (s, CH), 60.7 (s, CH ), 65.8 (s,
19
3
2
2
2
2
5
2
3
18
11
1
CH), 70.4 (s, CH), 70.9 (s, CH), 71.8 (s, CH), 77.8 (s,
C
q
), 79.0 (s,
q
C ), 96.5 (s, CH),
08.4 and 109.3 (s, 1
ppm] = 15.6 (s, 2B),
NH), 2979 (w,
3 and 14
C
q
), 156.1 (s,
10
C
q
). B{ H}-NMR (128 MHz, chloroform-d ):
11
1
δ
1
[
3
1
−
−
13.6 (s, 2B),
−
10.8 (s, 4B),
−
9.3 (s, 1B),
−
4.3 (s, 1B). IR (KBr):

̃
=
3
3
390 (w,
ν
ν
CH-sp ), 2933 (w,
ν
CH-sp ), 2594 (m,
ν
BH), 1707 (m, amide I),
CH), 1252 (m, amide III) cm . ESI-HRMS:
−
1
1
504 (m, amide II), 1455 (m,
δCH), 1366 (m,
δ
+
+
(
m/z) calculated for [C H B N O ] = 559.4386; observed 559.4395 [M+H] ; calculated
2
2
47 10
2
7
+
+
for [NaC H B N O ] = 581.4282; observed 581.4208 [M+Na] .
22
46 10
2
7
N-[(1,7-dicarba-closo-dodecaborane-1-yl)methyl]-N-(1,2:3,4-di-O-isopropylidene-6-deoxy-
galacatopyranos-6-yl)glycine ( ): Method A. Anhydrous TFA (4.20 mL, 54.4 mmol, 40.0 eq.)
was added dropwise under nitrogen atmosphere at 0 C to a solution of tert-butyl-N-[(1,7-
dicarba-closo-dodecaboran-1-yl)methyl]-N-(1,2:3,4-di-O-isopropylidene-6-deoxy- -D-galaca
topyranos-6-yl)glycinate ( ) (0.71 g, 1.36 mmol, 1.00 eq.) in 4.20 mL DCM. The mixture
α-D-
5
◦
α
3
was warmed to room temperature and stirred for 3 h. When the reaction was completed,
TFA and DCM were removed under reduced pressure. DCM and diethyl ether were used
as an entrainer to remove remaining TFA. The crude material was purified by column
chromatography using n-hexane/ethyl acetate (10:1, (v/v)) as eluent. Diethyl ether was

Molecules 2021, 26, 2057
10 of 16
used to remove remaining solvent. Compound
yield (300 mg, 0.63 mmol).
5
was obtained as a colorless solid in 47%
Method B. A Schlenk flask was charged with tert-butyl-N-[(1,7-dicarba-closo-dodecabor
an-1-yl)methyl]-N-(1,2:3,4-di-O-isopropylidene-6-deoxy- -D-galacatopyranos-6-yl)-glycina
te ( ) (0.50 g, 0.94 mmol, 1.00 eq.). Then, 4.0 mL anhydrous TFA (52.2 mmol, 55.6 eq.) were
α
3
added and the resulting solution was stirred at room temperature for 3 h. Afterwards,
about 4 mL DCM were added and both, TFA and DCM, were removed under reduced
pressure. This process was repeated three more times with DCM as an entrainer to remove
remaining TFA. Then, 3 mL concentrated NaHCO solution were added to the obtained
3
crude product while stirring and the mixture was subsequently sonicated for 15 min, re-
sulting in a thick, cloudy white suspension with a brownish oil-like layer on top. Upon
addition of 2 mL DCM, the oil-like layer was dissolved and gas evolution was observed.
Once the gas evolution had almost stopped, the solution was stirred for five more minutes.
Subsequently, the aqueous layer was removed using a syringe and extracted two times
with 2 mL ethyl acetate each. The combined organic layers were washed twice with 2 mL
of water. Afterwards, the combined organic layers were dried over MgSO and filtered.
4
Finally, the solvent was removed under reduced pressure, affording compound
yellowish foamy solid in 65% yield (290 mg, 0.612 mmol). H-NMR (400 MHz, chloroform-
5 as a
1
0
0
1
1,11 ,12 and 12
d1):
1
δ
[ppm] = 1.33, 1.34, 1.43 and 1.56 (s, 12H,
CH ), 1.50 to 3.00 (m, br, 10H,
3
6
15
13
2
0 BH), 2.91 to 2.99 (m, 3H, CH and CH), 3.17 (d, 1H, CHH, J
= 20.0 Hz), 3.21 (d,
2
HH
13
2
7
2
7
2
1
H, CHH, J
= 20.0 Hz), 3.48 (d, 1H, CHH, J
= 18.2 Hz), 3.59 (d, 1H, CHH, J
HH
HH
HH
5
4
3
3
=
18.2 Hz), 3.87 (m, 1H, CH), 4.15 (dd, 1H, CH, J
= 7.9 Hz, J
= 2.0 Hz), 4.35 (dd,
HH
HH
2
3
3
3
3
3
1H, CH, J
= 5.2 Hz, J
= 2,4 Hz), 4.62 (dd, 1H, CH, J
= 7.9 Hz, J
= 2.4 Hz),
HH
HH
1
HH
HH
3
13
1
5
2
(
.55 (d, 1H, CH, J
= 5.1 Hz). C{ H}-NMR (100 MHz, chloroform-d ):
δ
[ppm] = 24.4,
HH
1
0
0
1
1,11 ,12 and 12
6
15
7
4.8, 25.95 and 26.03 (s,
CH ), 54.6 (s, CH ), 55.3 (s, CH), 57.2 (s, CH ), 59.1
3
2
2
1
3
5
2
3
4
14
s, CH ), 65.5 (s, CH), 70.4 (s, CH), 70.8 (s, CH), 71.6 (s, CH), 76.3 (s,
C
q
), 96.4 (s,
2
1
9 and 10
8
11
1
CH), 108.8 and 109.7 (s,
[ppm] = 15.7 (s, 2B),
IR (KBr): = 3061 (w,
q q
1
C ), 172.4 (s, C ). B{ H}-NMR (128 MHz, chloroform-d ):
δ
−
−
13.5 (s, 2B),
−
11.3 (s, 2B),
−
10.6 (s, 2B),
−
9.0 (s, 1B),
−
4.5 (s, 1B).
2
3
3

̃
ν
CH -sp ), 2985 (w, νas.CH -sp ), 2961 (w, νs.CH -sp ), 2931 (m,
2
2
2
3
3
3
νas.CH -sp ), 2871 (w, νs.CH -sp ), 2595 (s,
δas.CH -sp ), 13 (m, δs.CH -sp ), 1066 (s,
ν
BH-sp ), 1716 (m,
ν
C=Ocarboxylic acid), 1456 (m,
3
3
3
3
-1
ν
C-O-Cether) cm . ESI-HRMS: (m/z) calculated
for [C H B NO ] = 474.3495; observed 474.3485 [M+H] .
N -[(1,7-Dicarba-closo-dodecaborane-1-yl)methyl]-N -(1,2:3,4-di-O-isopropylidene-6-deoxy-
-D-galactopyranos-6-yl)ethane-1,2-diamine ( ): A 25 mL round-bottom flask was charged
with 0.30 g (0.54 mmol, 1.00 eq.) tert-butyl-{2-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl-
1,2,:3,4-di-O-isopropylidene-6-deoxy- -D-galactopyranos-6-yl)amino]ethyl}-carbamate (
3
3
+
+
1
7
36 10
7
1
1
α
6
(
α
4)
and 2.00 mL (26.0 mmol, 48.2 eq.) TFA were added. The reaction mixture was stirred
for 3 h at room temperature. The reaction was stopped by adding 4 mL DCM with sub-
sequent evaporation of all volatile components under reduced pressure. This procedure
was repeated three times. The resulting crude product was further purified by adding
4
mL saturated NaHCO solution and sonication for about 15 min. Again, 3 mL DCM
3
were added with stirring, under observation of gas evolution, and after 5 min the resulting
layers were separated. The aqueous layer was extracted with 3 mL ethyl acetate. The
combined organic layers were washed twice with 2 mL distilled water each. The organic
layer was dried over MgSO , the drying agents were filtered off and the solvent was
4
removed under reduced pressure. Compound
hexane/ethyl acetate, 5:1, (v/v)) was isolated as a colorless foamy solid. H-NMR (400 MHz,
6
(0.25 g, 0.54 mmol, quant., R = 0.03, n-
f
1
0 0
2,12 ,13 and 13
1
chloroform-d1):
δ
[ppm] = 1.33, 1.34, 1.45, 1.54 (s, 12H,
CH ), 1.40 to 3.16 (m,
3
7
or 8
7 or/and 8
br, 10H, 10 BH), 2.62 to 2.69 (m, 1H,
CHH), 2.72 to 2.77 (m, 2H,
CHH), 2.77 to
7
or 8
CHH and CH ), 2.92 (s, 1H, CH), 3.00 (d, 1H, CHH, 2
6
16
14
2.86 (m, 3H,
J
= 15.4 Hz),
2
HH
.11 (d, 1H, 14CHH, 2JHH = 15.4 Hz) 3.87 (m, 1H, CH), 4.17 (dd, 1H, CH, JHH = 7.9 Hz
5
4
3
,
3
3
2
3
3
3
JHH = 1.9 Hz), 4.30 (dd, 1H, CH, J
= 5,2 Hz, J
= 2.4 Hz), 4.60 (dd, 1H, CH,
HH
HH
3J
= 7.9 Hz
,
3
JHH = 2.4 Hz), 5.52 (d, 1H, CH, J
1
3
= 5.1 Hz). C{ H}-NMR (100 MHz,
13
1
HH
HH
0
0
1
2,12 ,13 and 13
7 or 8
chloroform-d1):
δ
[ppm] = 24.5, 24.8, 25.96 and 26.0 (s,
CH ), 39.8 (s,
CH2),
3

Molecules 2021, 26, 2057
11 of 16
6
7 or 8
14
5
2
5
7
3.2 (s, CH ), 55.0 (s, 16CH), 57.7 (s,
CH ), 60.2 (s, CH ), 66.1 (s, CH), 70.3 (s, CH),
2
2
2
3
4
1
10 and 11
15
0.8 (s, CH), 72.0 (s, CH), 96.5 (s, CH), 108.5 and 109.3 (s,
C
q
),
C
q
was not ob-
11
1
servable (assumed at 77.7 ppm). B{ H}-NMR (128 MHz, chloroform-d ):
δ
[ppm] = 15.6
−
1
(s, 2B),
−
13.5 (s, 2B),
−
11.2 (s, 2B),
ν
−1
+
−
10.8 (s, 2B),
CH-sp ), 2593 (m,
CH-sp ) cm . ESI-HRMS: calculated for [C H B N O ] = 459.3862; ob-
−
9.3 (s, 1B),
BH-sp ), 1685 (w,
−
4.3 (s, 1B). IR (KBr):
NH-sp ), 1455 (w,

̃
= 2986
3
3
3
3
3
(w,
ν
CH-sp ), 2933 (w,
ν
δ
δ
CH-sp ),
3
+
1
381 (m,
δ
1
7
39 10
2
5
+
served 459.3851 [M+H] ; calculated for [C H B N O ] = 918.7724; observed 918.7607
34
77 20
4
10
+
[
2M+H] .
1
1
N -[(1,7-dicarba-closo-dodecaboran-1-yl)methyl]-N -(1,2:3,4-di-O-isopropylidene-6-deoxy-
α
-
2
D-galacatopyranos-6-yl)-N -[(4-methyl)-2-oxo-2H-chromen-7-yl)glycineamide (
7
): Method A.
Under nitrogen atmosphere, 1 mL (0.98 g, 12.4 mmol) of pyridine was added to a mix-
ture of N-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl]-N-(1,2:3,4-di-O-isopropylidene-6-
deoxy-α-D-galacatopyranos-6-yl)glycine (5) (100 mg, 0.21 mmol, 1.00 eq.) and 7-amino-4-
methylcoumarin (44.4 mg, 0.25 mmol, 1.20 eq.). After degassing the yellowish solution with
nitrogen, phosphorus(V) oxychloride (20.0
µ
L, 0.23 mmol, 1.10 eq.) was added dropwise
◦
at
−
18 C. The solution immediately changed to a red suspension and after a while back
◦
18 C. After the reaction was
to a yellow solution. The mixture was stirred for 1 h at
−
finished, the mixture was poured into H O and extracted with ethyl acetate. Subsequently,
2
the combined organic layers were washed with a NaHCO solution and a saturated NaCl
3
solution and then dried over MgSO . After filtration, the solvent was removed under
4
reduced pressure and the crude product was purified by column chromatography using
n-hexane/ethyl acetate (1:1, (v/v), R = 0.51) as eluent. Compound
7 was obtained as a
f
brownish solid in 27% yield (36 mg, 5.71 µmol).
Method B. N-[(1,7-dicarba-closo-dodecaboran-1-yl)methyl]-N-(1,2:3,4-di-O-isopropylid
ene-6-deoxy- -D-galacatopyranos-6-yl)glycine (86.1 mg, 0.18 mmol, 1.00 eq.) was added
α
to a Schlenk tube and was dissolved in 5 mL absolute DCM. Subsequently, the solution
◦
was cooled to 0 C in an ice bath and 7-amino-4-methylcoumarin (39.1 mg, 0.22 mmol,
1
.23 eq.), HOBt (29.5 mg, 0.22 mmol, 1.20 eq.), EDCI (45.1 mg, 0.24 mmol, 1.30 eq.) and
DIPEA (80 L, 0.46 mmol, 2.54 eq.) were added. Afterwards, the ice bath was removed, the
µ
reaction mixture was allowed to reach room temperature and stirred at room temperature
for 22 h. Subsequently, the solvent was removed under reduced pressure and the residue
was redissolved in 20 mL ethyl acetate and washed with 30 mL of 1 N HCl, concentrated
NaHCO and NaCl solution, respectively. The organic layer was dried over Na SO , fil-
3
2
4
tered and the solvent was removed under reduced pressure. The obtained crude product
was purified by column chromatography using n-hexane/ethyl acetate (1:1 (R = 0.51) to
f
1
:2, (v/v)) as eluent, affording compound
7
as a sticky, brownish solid in 18% yield (20 mg,
1
3
2
µ
mol). H-NMR (400 MHz, chloroform-d ):
δ
[ppm] = 1.28, 1.29, 1.38, 1.59 (s, 12H,
1
0
0
1
2,12 ,13 and 13
23
4
CH ), 1.50 and 3.00 (m, br, 10H, 10 BH), 2.42 (d, 3H, CH , J
= 1.2 Hz),
3
3
HH
6
6
16
14
2
2
(
.86 to 2.97 (m, 3H, CHH, CHH and CH), 3.16 (d, 1H, CHH, J
= 15.5 Hz), 3.25
HH
d, 1H, 14CHH, J
2
= 15.5 Hz), 3.43 (d, 1H, CHH, J
7
2
= 17.5 Hz), 3.52 (d, 1H, CHH,
7
HH
HH
2
5
4
3
JHH = 17.4 Hz), 3.93 (m, 1H, CH, J
= 9.7 and 2.5 Hz), 4.10 (dd, 1H, CH, J
= 7.8 Hz,
HH
HH
3
3J
= 2.0 Hz), 4.41 (dd, 1H, CH, J
2
3
= 5.2 Hz, J
3
= 2.4 Hz), 4.63 (dd, 1H, CH, J
3
=
HH
.8 Hz, J
HH
HH
HH
3
1
3
21
4
7
7
9
2
6
= 2.4 Hz), 5.69 (d, 1H, CH, J
= 5.2 Hz), 6.21 (d, 1H, CH, J
= 1.4 Hz),
HH
HH
HH
1
8
25
26
3
4
.53 to 7.58 (m, 2H, CH and CH), 7.71 (dd, 1H, CH, J
.57 (s, 1H, NH). C{ H}-NMR (100 MHz, chloroform-d ):
4.4, 24.7, 25.9 and 26.1 (s,
0.0 (s, CH ), 64.8 (s, CH), 70.3 (s, CH), 70.8 (s, CH), 71.7 (s, CH), 75.9 (s,
= 8.7 Hz, J
= 2.0 Hz),
HH
HH
9
13
1
23
δ
[ppm] = 18.6 (s, CH3),
1
0
0
1
2,12 ,13 or 13
6
16
14
CH ), 54.5 (s, CH ), 55.3 (s, CH), 58.7 (s, CH2),
3 2
7
5
2
3
4
15
q
C ), 96.5
2
1
18
10 and 11
21
26
(s, CH), 107.0 (s, CH), 108.9 and 109.6 (s,
q
C ), 113.4 (s, CH), 115.7 (s, CH),
16.2 (s, 2
2
C
q
), 125.2 (s, CH), 141.2 (s,
25
17
C
), 152.3 (s,
24
C
δ
), 154.3 (s,
[ppm] =
19
C
), 161.2 (s,
15.6 (s, 2B), 13.4 (s,
= 3288 (m,
20
1
1
2
q
q
q
q
q
C ),
8
11
1
69.3 (s, C
). B{ H}-NMR (128 MHz, chloroform-d ):
−
−
1
B), 11.2 (s, 2B),
−
−
10.5 (s, 2B),
−
8.9 (s, 1B),
−
4.4 (s, 1B). IR (KBr):

̃
ν
(NH,
2
3
3
secondary amide (trans)), 3058 (w,
2
ν
CH-sp ), 2985 (w, νasCH -sp ), 2957 (w, νsCH -sp ),
2
2
3
3
3
922 (m, νasCH -sp ), 2852 (m, νsCH -sp ), 2595 (m,
ν
BH-sp ), 1727 (s,
ν
C=Olactone), 1706
3
3
(
s, vC=Oamide, amide I), 1616 (
ν
C=C,
α
,ß-unsaturation), 1567 (νC=C, aromatic or δNH,

Molecules 2021, 26, 2057
12 of 16
3
amide II), 1519 (vC=C, aromatic or
ν
CN, amide II), 1454 (m, δasCH -sp ), 1369 (m, δsCH3-
3
3
2
2
sp ), 1066 (s,
ν
C-O-CEther), 901 (m,
δ
CH-sp /aromatic), 853, 806 (m,
δ
CH-sp /aromatic)
−
1
+
cm . ESI-HRMS: (m/z) calculated for [NaC H B N O ] = 653.3843; observed 653.3840
27
42 10
2
8
+
[
M+Na] .
2
5
N -(4-{[(2-Amino-4-hydroxypteridine-6-yl)methyl]amino}benzoyl)-N -{2-[(1,7-dicarba-
closo-dodecaboran-1-ylmethyl)-(1,2:3,4-di-O-isopropyliden-6-deoxy-
yl)amino]ethyl}-L-glutamine ( ) and (S)-2-(4-{[(2-amino-4-hydroxypteridine-6-yl)methyl]am
ino}benzamido)-N ,N -bis-{2-[(1,7-dicarba-closo-dodecaborane-1-yl)methyl-(1,2:3,4-di-O-iso
propylidene-6-deoxy- -D-galactopyranos-6-yl)amino]ethyl}amino)pentanediamide ( ): A
00 mL Schlenk flask was charged with 0.24 g (0.55 mmol, 1.00 eq.) folic acid and 20 mL
α-D-galactopyranos-6-
8
1
5
α
9
1
dimethylformamide were added. The mixture was sonicated for 15 min and, subse-
◦
quently, warmed to 37 C for 15 min until a clear solution was obtained. To this mix-
ture, 0.11 g (0.55 mmol, 1.00 eq.) DCC and 0.06 g (0.55 mmol, 1.00 eq.) NHS were
added. The reaction mixture was stirred for 16 h at room temperature. Afterwards, 0.25 g
1
1
(
0.55 mmol, 1.00 eq.) N -[(1,7-dicarba-closo-dodecaborane-1-yl)methyl]-N -(1,2:3,4-di-O-
isopropylidene-6-deoxy- -D-galactopyranos-6-yl)ethane-1,2-diamine ( ), dissolved in 7 mL
α
6
dimethylformamide, were added to this solution and the mixture was stirred overnight
at room temperature. The resulting suspension was filtered under inert conditions. Sub-
sequently, 0.10 mL (0.61 mmol, 1.10 eq.) N,N-diisopropylethylamine were added and
the mixture was stirred overnight at room temperature. The reaction was stopped by
adding 50 mL ice-cold diethyl ether. Simultaneously, the mixture was cooled in an ice
bath. Completion of the precipitation was achieved by storage for one additional night
◦
at
−
20 C. The resulting precipitate was filtered off and washed with 10 mL ice-cold
diethyl ether. The precipitate was suspended in 5 mL diethyl ether and subsequently
sonicated for 15 min. Afterwards, the raw product was filtered again. The orange-red
solid was washed with 5 mL diethyl ether and the previously described procedure was
repeated one more time. Afterwards, the precipitate was dried in vacuo. It was not pos-
sible to isolate the desired compound
of N -(4-{[(2-amino-4-hydroxypteridine-6-yl)methyl]amino}benzoyl)-N -{2-[(1,7-dicarba-
8
, but mass spectrometry revealed the presence
2
5
closo-dodecaboran-1-yl)methyl-(1,2:3,4-di-O-isopropyliden-6-deoxy-
yl)amino]ethyl}-L-glutamine ( ) and (S)-2-(4-{[(2-amino-4-hydroxypteridine-6-yl)methyl]am
ino}benzamido)-N ,N -bis-{2-[(1,7-dicarba-closo-dodecaborane-1-yl)methyl-(1,2:3,4-di-O-iso
propylidene-6-deoxy- -D-galactopyranos-6-yl)amino]ethyl}amino)pentanediamide ( ). Due
α-D-galactopyranos-6-
8
1
5
α
9
to impurities, it was not possible to determine a yield based on HRMS.
8
: ESI-HRMS:
+
+
(
m/z) calculated for [C H B N O ] = 883.5139; observed 883.5151 [M+H] ; calcu-
36
56 10
+
9
10
+
lated for [NaC H B N O ] = 905.4959; observed 905.4963 [M+Na] ; calculated for
[
3
6
55 10
+
9
10
+
KC H B N O ] = 921.4700; observed 921.4715 [M+K] .
9
ν
:1E7D IR (KBr):

̃
= 3315
3
ν
6
55 10
3
9
10
3
3
3
(
w,
NH-sp ), 2931 (w,
ν
CH-sp ), 2850 (w,
ν
CH-sp ), 2594 (w,
BH-sp ), 1723 (m, amide
3
−1
I), 1687 (m, amide II), 1605 (s,
calculated for [C H B N O ] = 1323.8830; observed 1323.8829 [M+H] ; calculated
for [NaC H B N O ] = 1345.8649; observed 1345.8625 [M+Na] ; calculated for
ν
C=Carom.), 1412 (m,
δ
CH-sp ) cm . ESI-HRMS: (m/z)
+
+
5
3
92 20 11 14
+
+
53
91 20 11 14
+
+
[
KC H B N O ] = 1361.8390; observed 1361.8349 [M+K] .
53 91 20 11 14
4. Conclusions
In this work we reported the successful design of a novel modular, small-molecule-
based approach to synthesizing boron-rich compounds bearing a carboxylic acid group
or a primary amine group as potential coupling partners for suitable tumor-selective
biomolecules. As proof of concept, conjugates with 7-amino-4-methylcoumarin and folic
acid were obtained. While the present work focused on the development of a synthetic
protocol, the next steps will include the deprotection of the respective galactopyranosyl
protecting groups under acidic aqueous conditions [61] followed by biological investiga-
tions.
Supplementary Materials: Supplementary information is available online, including the numbering
scheme of the isolated compounds 1-7, NMR spectra of compounds 5, 6 and 7 and mass spectra

Molecules 2021, 26, 2057
13 of 16
0
0
, ESI-4 and 1-
of
8
and
9, additional synthetic procedures and analytical data for
2
,
ESI-3
,
ESI-3
(
trifluoromethanesulfonylmethyl)-1,7-dicarba-closo-dodecaborane(12), crystallographic information
0
for compound ESI-3 , information about the exploration of the optimization for the synthesis of
3
and the deprotection protocol for 4, and the extension of the synthetic protocol to ortho-carborane
derivatives.
Author Contributions: Conceptualization, M.K. and E.H.-H.; Data curation, M.K. and P.L.; formal
analysis, M.K., J.-S.J.F., A.F., N.A.U., J.T. and P.L.; funding acquisition, E.H.-H.; investigation, M.K.,
J.-S.J.F., A.F., N.A.U. and J.T.; methodology, M.K.; project administration, M.K. and E.H.-H.; resources,
E.H.-H.; supervision, E.H.-H.; validation, M.K., J.-S.J.F., A.F., N.A.U. and J.T.; visualization, M.K.;
writing—original draft, M.K.; writing—review & editing, J.-S.J.F., A.F., N.A.U., J.T., P.L. and E.H.-H.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Europäischer Fonds für regionale Entwicklung (EFRE),
the Free State of Saxony (ESF) and the Leipzig School of Natural Sciences—Building with Molecules
and Nano-objects (BuildMoNa).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We thank Ramona Oehme, Susann Billig and Claudia Birkemeyer for measuring
the mass spectra and Ines Rein, Stefanie Märcker-Recklies and Jaqueline Lewandowski for recording
the infrared spectra.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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