2,3-Diaminopropanols obtained from D-serine as intermediates in the synthesis of protected 2,3-L-diaminopropanoic acid (L-DAP) methyl esters

Article abstract of DOI:10.3390/molecules25061313

A synthetic strategy for the preparation of two orthogonally protected methyl esters of the non-proteinogenic amino acid 2,3-l-diaminopropanoic acid (l-Dap) was developed. In these structures, the base-labile protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was paired to the p-toluensulfonyl (tosyl, Ts) or acid-labile tert-butyloxycarbonyl (Boc) moieties. The synthetic approach to protected l-Dap methyl esters uses appropriately masked 2,3-diaminopropanols, which are obtained via reductive amination of an aldehyde prepared from the commercial amino acid N^α-Fmoc-O-tert-butyl-d-serine, used as the starting material. Reductive amination is carried out with primary amines and sulfonamides, and the process is assisted by the Lewis acid Ti(OⁱPr)₄. The required carboxyl group is installed by oxidizing the alcoholic function of 2,3-diaminopropanols bearing the tosyl or benzyl protecting group on the 3-NH₂ site. The procedure can easily be applied using the crude product obtained after each step, minimizing the need for chromatographic purifications. Chirality of the carbon atom of the starting d-serine template is preserved throughout all synthetic steps.

Full text of DOI:10.3390/molecules25061313

molecules
Article
2
,3-Diaminopropanols Obtained from d-Serine as
Intermediates in the Synthesis of Protected
,3-l-Diaminopropanoic Acid (l-Dap) Methyl Esters
2
1
2
2
Andrea Temperini , Donatella Aiello
, Fabio Mazzotti ,
Constantinos M. Athanassopoulos 3 , Pierantonio De Luca 4 and Carlo Siciliano 5,*
1
Dipartimento di Scienze Farmaceutiche, Università di Perugia, Via del Liceo 1, 06123 Perugia, Italy;
andrea.temperini@unipg.it
Dipartimento di Chimica e Tecnologie Chimiche (CTC), Via Ponte P. Bucci, Cubo 12D, Università della
2
Calabria, I-87036 Arcavacata di Rende (CS), Italy; donatella.aiello@unical.it (D.A.);
fabio.mazzotti@unical.it (F.M.)
Department of Chemistry, University of Patras, GR-26504 Patras, Greece; kath@chemistry.upatras.gr
3
4
Dipartimento di Ingegneria Meccanica, Energetica e Gestionale, Università della Calabria, I-87036
Arcavacata di Rende (CS), Italy; pierantonio.deluca@unical.it
Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Edificio Polifunzionale, Universit
Calabria, I-87036 Arcavacata di Rende (CS), Italy
5
à della
*
Correspondence: carlo.siciliano@unical.it; Tel.: +39-0984-493192
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: M. Graça P. M. S. Neves and Gianfranco Favi
Received: 22 February 2020; Accepted: 12 March 2020; Published: 13 March 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: A synthetic strategy for the preparation of two orthogonally protected methyl esters of
the non-proteinogenic amino acid 2,3-l-diaminopropanoic acid (l-Dap) was developed. In these
structures, the base-labile protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was paired to
the p-toluensulfonyl (tosyl, Ts) or acid-labile tert-butyloxycarbonyl (Boc) moieties. The synthetic
approach to protected l-Dap methyl esters uses appropriately masked 2,3-diaminopropanols,
which are obtained via reductive amination of an aldehyde prepared from the commercial amino
α
acid N -Fmoc-O-tert-butyl-d-serine, used as the starting material. Reductive amination is carried
i
out with primary amines and sulfonamides, and the process is assisted by the Lewis acid Ti(O Pr) .
4
The required carboxyl group is installed by oxidizing the alcoholic function of 2,3-diaminopropanols
bearing the tosyl or benzyl protecting group on the 3-NH site. The procedure can easily be
2
applied using the crude product obtained after each step, minimizing the need for chromatographic
purifications. Chirality of the carbon atom of the starting d-serine template is preserved throughout
all synthetic steps.
Keywords: 2,3-l-diaminopropanoic acid; l-Dap; basic amino acids; serine; reductive amination;
orthogonal protecting groups; 1,3,5-trichloro-2,4,6-triazinetrione; trichloroisocyanuric acid (TCCA);
(2,2,6,6-tetramethylpiperidi-1-yl)oxyl (TEMPO); N-methylation
1
. Introduction
1
.1. The Role of l-Dap in Natural and Synthetic Compounds
2
,3-l-Diaminopropanoic acid (l-Dap) is a non-proteinogenic amino acid produced by numerous
plants and bacteria [ ]. It is the main structural motif of albizziine, a natural non-proteinaceous
free amino acid which is ubiquitously present in higher plants and in seeds [ ]. The known
preparative methods for the total synthesis of albizziine employs protected derivatives of l-Dap
as the starting materials [ ]. Bleomycins (BLMs) are structurally related glycopeptide-derived
1
2
3
Molecules 2020, 25, 1313; doi:10.3390/molecules25061313
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1313
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antitumor antibiotics, and they display a l-Dap amide common scaffold as the chain N-terminus [4–8].
l-Dap is also one of the most frequently found amino acids inserted in bioactive peptides, such as
cyclotheonamide B, phleomycin, paenibacterin derivatives, lavendomycin, gastrin-releasing peptide
receptor-specific scaffolds, capreomycin, various peptide-based antibacterials, and peptidomimetics,
such as vitronectin [9–16]. Protected l-Dap derivatives were proposed as powerful inhibitors of the
formation of advanced glycosylation end-products (AGEs), which are implicated in the development
of diabetes mellitus and neurodegenerative Alzheimer’s and Parkinson’s diseases [17]. Advanced lipid
peroxidation end-product (ALE) formation can also be modulated by the same l-Dap derivatives used to
control AGE cascades, against the endogenous peroxidation of polyunsaturated fatty acids [17]. Matrix
metalloproteases, containing l-Dap as the terminal carboxylate amino acid unit, were synthesized and
biologically tested for their roles in the degradation and remodeling of connective tissues [18]. l-Dap
is contained in staphyloferrin B, as well as in a series of synthetic and natural siderophores [19,20].
Appropriately protected l-Dap subunits were used as building blocks in the solid-phase synthesis of
chelating peptide conjugates having therapeutic and diagnostic applications in tumor diseases [21].
Dismutation of superoxide species can be controlled by metal complexes of l-Dap branched peptides
with divalent cations [22,23]. l-Dap dipeptides were used as reference structures to investigate
the ionizability of side-chain primary amino functions in amino acids [24], and the substitution of
aspartic units by l-Dap was adopted in the synthetic preparation of glycoproteins [25]. l-Dap acts
as crosslinker in graphene sponges of clinical importance for hemostasis control [26], and it is a
component of biosynthetic model compounds employed in studies aiming to define acyl-enzyme
activities and foldings [27
,28]. l-Dap-based peptide dendrimers catalyze asymmetric processes in
organic synthesis [29 30]. Furthermore, l-Dap-enriched peptide backbones were obtained for gene
,
α
β
silencing scopes [31], and synthetic orthogonally N ,N -diprotected l-Dap-based nucleoamino acids
were investigated for the assembly of cationic nucleopeptides in biomedical applications [32]. Recently,
the identification of l-Dap in the Murchison chondritic meteorite [33] suggested the possible role of this
non-proteinogenic amino acid as a selective molecular target to investigate prebiotic polycondensation
processes of importance in establishing the appearance of life on Earth [34].
1
.2. Synthetic Routes to l-Dap
The last decade was characterized by the disclosure of a wide range of synthetic routes to l-Dap
and its analogues. Much effort was spent searching for convenient methods which could employ
simple molecular scaffolds [35]. Natural amino acids were proposed as convenient starting materials;
however, the use of serine was seldom investigated [36–39]. A combination of the classical series of acid-
and base-labile masking groups used in the solution and solid-phase peptide synthesis was generally
found advantageous. The installation of two or more amino functions in natural carbon backbones
and their heterocycle analogues might present additional difficulty [40]. Another critical step is the
preservation of the absolute configuration of chiral carbon atoms when molecules from the natural
α
β
chiral pool might be used as enantiomerically pure starting materials. Differently N ,N -diprotected
l-Dap, as well as its mono- and di-alkylated derivatives, was obtained through a plethora of synthetic
approaches [41–52]. In general, the published methods are characterized by variable overall yields
in l-Dap, and they are notable for needing several steps of chromatography and for the problematic
stereochemical control of reactions.
2
. Scope of the Work
The scope of the present work was an investigation of a preparative route to appropriately
protected l-Dap methyl esters, which may further be employed in branching, alkylation, cyclization,
cross-linking, and solid-phase peptide synthesis. More specifically, we were attracted by the design of
an advantageous synthetic procedure that might include (i) the easy replacement of protections placed
on the amino functions of l-Dap, (ii) the methylation of the carboxyl function to obtain l-Dap methyl

Molecules 2020, 25, 1313
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esters, and (iii) the possible use of appropriate masking groups which could enable a chemoselective
β
N -methylation, in order to prepare suitable scaffolds for the elaboration of alkylated l-Dap derivatives.
In particular, our synthetic plan was chalked out starting from optically pure protected d-serine.
i
The key synthetic step of the procedure was the Ti(O Pr) -assisted reductive amination [53
–
55] of a
4
protected d-serine aldehyde derivative to give a series of protected 2,3-diaminopropanols, which were
oxidized under mild conditions [56,57]. It is worth noting that d-serine was used to obtain the correct
l-Dap stereochemistry.
3
. Results and Discussion
3
.1. Retrosynthetic Analysis
α
β
Scheme 1 shows the retrosynthetic analysis for obtaining N ,N -diprotected l-Dap methyl esters.
Enantiomerically pure Fmoc-O-tert-butyl-d-serine (1), commercially available at low cost, was selected
as the ideal precursor of the targeted compounds.
α
β
Scheme 1. The retrosynthetic analysis for the preparation of orthogonally N ,N -diprotected
1
2
2
,3-l-diaminopropanoic acid (l-Dap) methyl esters (PG and PG are orthogonal groups).
It was thought feasible to realize the desired 2,3-diamino framework via the synthesis of suitable
intermediates having a 2,3-diaminopropanol scaffold, prepared via reductive amination of a d-serine
aldehyde derivative with different primary amines and arylsulfonamides, bearing protecting groups
compatible with the conditions of the solution and solid-phase peptide chemistry. The mild oxidation
of alcohol group of the amine-protected 2,3-diaminopropanols could generate the carboxylic group
required to complete the 2,3-diamino acid backbone. Finally, the carboxyl function can easily be
methylated to afford the desired fully protected l-Dap methyl esters. The plan was designed in order to
1
protect the 2-NH group of the methyl esters with the base-labile Fmoc group (PG ). In that structure,
2
the 3-NH amino function could orthogonally be protected to give a tosyl derivative, or one with an
2
2
acid-labile Boc group (PG ). In the 2,3-diamminopropanol intermediates, the simultaneous presence of
a benzyl moiety and an orthogonal Fmoc protection on the 3- and 2-NH functions, respectively, could
2
also support the facile interconversion of the pair of amino protections, enabling the installation of Boc
1
2
(
PG ) and Fmoc (PG ) groups at the 2- and 3-positions of the final l-Dap methyl ester structures.
3
.2. Synthesis of the 2,3-Diaminopropanols
Scheme 2 displays our synthetic route to the Fmoc-protected 2,3-diaminopropanol intermediates
4
–
10. These compounds were obtained via reductive amination of
reacted with N,O-dimethylhydroxylamine hydrochloride [58] to give the
, which was recovered in a 94% yield, without need for chromatographic
purification. Reduction of by LiAlH afforded the corresponding -amino aldehyde , which was
obtained in a 92% yield, avoiding chromatography. The structures of compounds and were
was then subjected to the reductive
3 with amines and sulfonamides.
The protected amino acid
Weinreb–Nahm amide
1
2
2
α
3
4
2
3
1
13
confirmed by H- and C-NMR spectroscopy. Compound
3
amination with a series of primary amines and sulfonamides having different structural characteristics.
Benzylamine was chosen as an ideal model to check the reaction performance. The process was
i
assisted by the Lewis acid Ti(O Pr) , in the presence of sodium cyanoborohydride, a reducing agent
4
which is widely employed in metal-assisted reactions of amino acids and their derivatives [59,60].

Molecules 2020, 25, 1313
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Primary amines result in the removal of the base-labile N-masking Fmoc group; however, the use of a
Lewis acid prevents this practical inconvenient [61]. Compound reacted smoothly with benzylamine,
in the presence of the titanium(IV) species and the hydride donor, to afford
3
4
in excellent yields (92%),
1
after chromatography. Thin-layer chromatography (TLC) analysis, as well as the H-NMR spectrum of
the crude reaction material, highlighted the absence of unwanted by-products generated through the
removal of the base-labile Fmoc protecting group.
Table 1. Reductive amination of 3: synthesis of the 2,3-diaminopropanol intermediates.
Reagent
Product
2,3-Diaminopropanol Structure
Yield (%) 1
4
92
5
90
6
7
8
90
91
89
9
82
85
1
0
1
Yields in final products obtained after chromatography.

Molecules 2020, 25, 1313
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Scheme 2. Synthesis of Fmoc-2,3-diaminopropanols
Weinreb–Nahm amide , (a) 1-hydroxybenzotriazole (HOBt) monohydrate/N,N -diisopropylcarbodiimide
DIC)/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 2 h, room temperature
r.t.); then, N,O-dimethylhydroxylamine hydrochloride/DIEA/DCM, overnight, r.t. (94% yield);
4–10. Experimental conditions: preparation of the
0
2
(
(
preparation of the Garner-like aldehyde
amination, (c) amine or arylsulfonamide/Ti(O Pr) /EtOH, 10 min, r.t., then NaBH CN, overnight, r.t.
3
, (b) LiAlH /THF, 12 min, r.t. (92% yield); reductive
4
i
4
3
(82%–92%, Table 1).
The molecular structure of
spectroscopy. In particular, the 1D H- and C-NMR spectral plots displayed the correct number,
multiplicity, and integral values of all resonances assignable to the expected structure of . The contour
plot of the 2D homonuclear proton TOCSY spectrum of the benzyl derivative did not show signal
overlap, facilitating the recognition of a set of cross-peaks at 5.52 ppm, in the regions between 3.90 and
.40 ppm, and between 2.60 and 2.90 ppm, which were generated by the resonances of all correlated
protons along the molecular backbone of (Figure 1). Two-dimensional spectroscopy unequivocally
4 was confirmed by one- and two-dimensional (1D and 2D) NMR
1
13
4
4
3
4
proved the successful installation of the second amino function in substitution of the hydroxyl group
of the starting d-serine backbone. These results highlighted that the reductive amination process can
be a useful strategy for the realization of orthogonally protected 2,3-diaminopropanols.
ppm
1
2
.5
.0
Current Data Parameters
NAME
MARTA CM16 grezzo TOCSY
EXPNO
PROCNO
1
1
F2
-
Acquisition Parameters
20130223
Date_
Time
22.03
INSTRUM
PROBHD
PULPROG
TD
AV300
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
5
mm BBO BB-1H
mlevph
512
SOLVENT
NS
CDCl3
64
DS
SWH
16
CH NHCH Ph
2 2
4496.403 Hz
FIDRES
AQ
8.782037 Hz
0.0569844 sec
2048
RG
DW
111.200 usec
6.00 usec
DE
TE
300.0 K
d0
0.00000300 sec
D1
2.50000000 sec
D9
0.08000000 sec
d12
0.00002000 sec
FACTOR1
IN0
6
0.00022240 sec
l1
36
CH O + CH Ph
MCREST
MCWRK
SCALEF
ST1CNT
0.00000000 sec
2
2
1.25000000 sec
6
0
=
======= CHANNEL f1 ========
CHCH NH
2
NUC1
P1
1H
6.80 usec
p5
23.34 usec
35.00 usec
70.00 usec
2500.00 usec
-3.00 dB
-1.00 dB
300.1316681 MHz
P6
p7
P17
PL1
PL10
SFO1
F1
-
Acquisition parameters
ND0
TD
1
256
SFO1
300.1317 MHz
FIDRES
SW
17.564074 Hz
14.981 ppm
States-TPPI
FnMODE
F2
-
Processing parameters
SI
1024
SF
300.1300000 MHz
WDW
SSB
LB
SINE
2
0.00 Hz
0
CHCH NH
GB
PC
2
1.40
F1
-
Processing parameters
SI
1024
MC2
SF
States-TPPI
300.1300000 MHz
NHFmoc
WDW
SSB
LB
SINE
2
0.00 Hz
0
GB
CH O + CH Ph CH NHCH Ph
2
2
2
2
6
7
7
8
8
9
.5
.0
.5
.0
.5
.0
9
.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm
1
1
Figure 1. Contour plot of the two-dimensional (2D) homonuclear H, H-TOCSY spectrum recorded
for compound 4.

Molecules 2020, 25, 1313
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The more sterically demanding diphenylmethylamine (benzhydrylamine) was a probe for
observing the possible steric effects exerted by the nucleophile, as well as to prepare a
2
,3-diaminopropanol scaffold containing the acid-labile benzhydryl masking group, which is orthogonal
to the Fmoc protection [62]. Treatment of with benzhydrylamine afforded the corresponding protected
,3-diaminopropanol , which was obtained in a 90% yield after chromatography. Results highlighted
that the nucleophile size did not modify yields and reaction times. The proton NMR spectrum of
3
2
5
5
showed signal duplication, line broadening, and overlapping. Spectral data suggested the possible
presence in solution of two main set of conformers in a 75:25 ratio, at the temperature fixed for NMR
experiments. Coexistence of the two conformers is likely due to the slow rotation (on the NMR
time-scale) around the amide bond of the carbamate residue. In carbamates, the two conformers syn
and anti undergo dynamic exchange, with a general predominance of the anti isomer conformation
with respect to its syn counterpart due to steric and electrostatic effects that can change the dipole
moment and bond angles [63
difference between the syn and anti conformers [65], and intra- and intermolecular hydrogen bonding
may also perturb the syn/anti equilibrium [66]. In the case of compound , the minor conformer was
,64]. Solvent, temperature, and concentration can affect the free energy
5
observable since it could be stabilized by the size and electronic effects of the benzhydryl group, as well
as by the NMR solvent that could remarkably influence transition state energies and the rotational
barrier in the syn/anti conformer exchange [67].
Alkylation of the amino functions is one of the most interesting modifications of l-Dap,
and methylation of the 3-NH group is a structural feature displayed by many biologically active
2
compounds [68
,
69]. Reductive amination of
9% yield after chromatography. The reaction represented a short route to the direct alkylation of the
-NH function of the 2,3-diaminopropanol scaffold, and the n-propyl derivative was considered to
3 with n-propylamine (Scheme 1, Table 1) gave 8 in an
8
3
8
2
be a possible precursor in the synthesis of alkylated l-Dap structures.
3
.3. Analysis of Racemization
Chiral -amino aldehydes show the tendency to racemize under acidic or basic conditions,
α
as well as during chromatographic purification over silica gel [70]. In order to check whether the
chiral carbon atom configuration of the starting d-serine is preserved during the reductive amination
process, the
α
-amino aldehyde
3
was reacted with the enantiomerically pure (S) and (R) isomers of
and
1
-methylphenylamine. Reactions gave the corresponding diastereomeric 2,3-diaminopropanols
6
7
(Scheme 2, Table 1), which were obtained in a 90% and 91% yield, respectively, after chromatography.
1
Both pure compounds
6
and
7
exhibited the expected molecular structure, as established by H- and
13
C-NMR analysis and MS experiments. The NMR analysis was extended to the respective crude
materials, as obtained from the reaction work-up, avoiding further purification steps. In this case,
each NMR spectrum displayed signals which were diagnostically correlated to the proton and carbon
resonances of a single diastereomer, with no evidence for signals attributable to other diastereomers,
at least under the sensitivity limits of the spectrometric technique. Spectral data indicated that the
Ti(IV)-assisted process of the imine intermediates was stereospecific [71]. Moreover, the absence of
detectable resonance signals due to diastereomeric mixtures in each crude sample suggested that the
reductive amination proceeded with no change of the configuration of the chiral carbon atom of
3,
consequently preserving the stereochemistry of the starting amino acid . The absence of racemization
1
was definitively confirmed by reverse-phase (RP) U-HPLC/MS analysis. Chromatograms recorded for
separate injections of crude 6 and 7 showed a single elution peak for each compound (Figure 2).

Molecules 2020, 25, 1313
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1
00
8.80
9
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
100
95
90
85
80
8.53
(A)
(C)
75
70
65
60
55
50
8.49
1.88
2
10.37
5
0
0
1
3
4
5
6
7
8
9
10
11
12
13
14
Time (min)
4
5
1
00
8.51
40
35
30
25
20
15
8.88
9
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
(B)
10.21
12.40
1.88
1.76
12.04
10
10.36
10.43
5
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (min)
1.75
5
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (min)
Figure 2. Reverse-phase (RP) U-HPLC/MS analysis of crude
diastereomers 6 and 7 (C).
6
(
A
), crude
7
(B
), and a 2:1 mixture of
3
.4. Reductive Amination of the Aldehyde 3 with Sulfonamides
We focused our attention on the reductive amination of
-nitrobenzenesulfonamide (Scheme 2, Table 1). The required nucleophiles were prepared from the
3
with p-toluenesulfonamide and
4
corresponding commercial sulfonyl chlorides, via reaction with a 20% ammonia solution. Reductive
amination of produced the arylsulfonyl-protected 2,3-diaminopropanols and 10, in very high yields
82% and 85%, respectively), after column chromatography. NMR spectroscopy and mass spectrometry
experiments confirmed the structures of both compounds. The successive treatment of with a slight
excess of trimethyloxonium tetrafluoroborate [72 73] was site-specific, furnishing 11 as the unique
product of methylation, in a quantitative yield, and without need for chromatography (Scheme 3).
3
9
(
9
,
O
NO2
O
NO2
H O
N
O
S
Fmoc
Fmoc
N
N
S
N
H
H
O
O
9
11
Scheme 3. Methylation of
quantitative yield).
9.
Experimental conditions: (CH O) BF /DIEA/DCM, 10 min, r.t.
3 3 4
(
Alternatively, methylation of
9 was realized using a threefold excess of diazomethane in
dichloromethane, at room temperature. In this case, compound 11 was obtained, in short times,
in quantitative yields, and avoiding chromatography. However, for a gram-scale preparation,
diazomethane might be not the best option, because of its toxicity and dangerous preparation [74].
β
Compound 11 was reputed to be a possible intermediate for the synthesis of N -methylated l-Dap
scaffolds, after transformation of the OH function into a carboxyl group, followed by the final removal
of the arylsulfonyl protection.
3
.5. Synthesis of the l-Dap methyl Ester 14
The 2,3-aminopropanol 10 featured three orthogonal masking groups, a favorable characteristic for
completing the l-Dap backbone realization. The presence of a tosyl group, which is largely employed
in amino acid chemistry and peptide synthesis [75 76], convinced us to select 10 as the most likely
,
candidate for the development of a short synthetic route to the desired l-Dap backbone. We designed an
economical and mild method, which can limit chromatography and preserve sensitive masking groups.

Molecules 2020, 25, 1313
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The synthetic procedure is depicted in Scheme 4. Initially, the 2,3-diaminopropanol 10 was
reacted with trifluoroacetic acid (TFA) in the presence of trifluoroethanol (TFE). Acidolysis of
the tert-butyl ether moiety generated the 2,3-diaminopropanol 12, which was quantitatively
obtained without need for chromatography. Compound 12 was then subjected to oxidation with
1
,3,5-trichloro-2,4,6-triazinetrione; trichloroisocyanuric acid (TCCA) in the presence of catalytic amounts
of (2,2,6,6-tetramethylpiperidi-1-yl)oxyl (TEMPO) and sodium bromide, to afford 13. The crude
carboxylic acid was finally treated with diazomethane in dichloromethane at room temperature, to give
the l-Dap methyl ester 14, which was obtained in an excellent overall yield, without chromatography.
O
OH
H O
O
OH
H O
H O
N
Step 1
Step 2
Fmoc
Fmoc
N
Fmoc
N
N
S
N
S
N
H
S
H
H
O
O
O
10
12
13
Step 3
Cl
N
O•
N
O
O
TEMPO =
TCCA =
N
N
O
O
Cl
Cl
H O
N
O
Fmoc
2,2,6,6-Tetramethylpiperidin-1-yl)oxyl
Trichloroisocyanuric acid
N
H
S
O
14
Scheme 4. Oxidation of 10 with; 1,3,5-trichloro-2,4,6-triazinetrione (trichloroisocyanuric acid,
TCCA)/ (2,2,6,6-tetramethylpiperidi-1-yl)oxyl (TEMPO): preparation of Fmoc-l-Dap(Ts)-OCH (14
)
3
Experimental conditions: Step 1 (acydolysis), Trifluoroacetic acid (TFA)/Trifluoroethanol (TFE)/DCM,
5 min, r.t. (quantitative yield); Step 2 (oxidation), TCCA/TEMPO/NaBr/aq. NaHCO /Acetone, 20 min.
1
3
◦
at 0 C, then overnight, r.t.; Step 3 (methylation), 0.66 N CH N in DCM, 15 min, r.t. (14, yields: 92%
2
2
over 2 steps, and 68% referred to the initial amount of 1).
3
.6. Synthesis of the l-Dap Methyl Ester 21
Continually, we investigated the applicability of the TEMPO-mediated oxidation to the preparation
of a l-Dap methyl ester, bearing the acid-sensitive Boc and the base-labile Fmoc group on the 2- and
-NH functions, respectively. We were attracted by the idea of masking the side-chain amino function
3
2
of l-Dap with the Fmoc group, because this protection can easily be removed under mild conditions
during amino acid coupling sequences in solution phase and/or on solid supports, generally used in the
total synthesis of oligopeptide strands and biologically active peptides. To fulfill our aim, we turned
our attention on the benzyl-protected 2,3-diaminopropan-1-ol 4. The presence of the benzyl group is
beneficial, because it can effortlessly be removed by catalytic hydrogen transfer, preserving sensitive
functional groups, base- and acid-labile masking moieties, and the chiral integrity of neighboring
chiral carbon atoms. We planned a multi-step procedure, which could sequentially employ only crude
products, as they are obtained via the simple work-up of the respective reactions, minimizing the
number of time-expending and tedious chromatographic purifications. Scheme 5 displays the synthetic
approach used to convert 4 into the targeted fully protected l-Dap methyl ester 21.

Molecules 2020, 25, 1313
9 of 21
Scheme 5. Multistep synthesis of Boc-l-Dap(Fmoc)-OH (20) and its derivative Boc-l-Dap(Fmoc)-OCH3
21). Experimental conditions: Step 1 (acidolysis), TFA/TFE/DCM, 15 min, r.t.; Step 2 (oxidation),
(
◦
TCCA/TEMPO/NaBr/aq. NaHCO /Acetone, 20 min at 0 C, then overnight, r.t.; Step 3 (Fmoc removal),
3
2
0% DEA in DCM, 30 min, r.t.; Step 4 (protection with Boc), Boc O/H O/1,4-dioxane/NaHCO , overnight,
2 2 3
◦
r.t.; Step 5 (hydrogenolysis), 1,4-cyclohexene/10% Pd-C/EtOH, overnight, 25 C; Step 6 (protection
with Fmoc), Fmoc-OSu/H O/1,4-dioxane/NaHCO , overnight, r.t.; Step 7 (methylation), 0.66 N CH N
2
3
2
2
in DCM, 15 min, r.t. (Yields: 20, 93% over 6 steps, and 73% referred to the initial amount of
quantitative from 20).
1
;
21
,
The conversion of
. Unlike the synthesis of methyl ester 14, we preferred to directly oxidize the crude intermediate
,3-diaminopropanol 15, without further purification. The reagent system TCCA/TEMPO/NaBr was
used to obtain 16, which was in turn treated with diethylamine (DEA) in dichloromethane, to remove the
4 into 20 began with the acidolysis of the tert-butyl ether linkage, to generate
1
5
2
Fmoc protecting group. Unblocking of the 2-NH function afforded the carboxylic acid 17, which was
2
subsequently reacted with di-tert-butyl dicarbonate (Boc O) to yield the Boc-derivative 18. A catalytic
2
transfer hydrogenation step [77] was applied to remove the benzyl residue from the 3-NH function of 18
,
2
and the obtained carboxylic acid 19 was reacted with (N-(9-fluorenylmethoxycarbonyloxy)succinimide)
Fmoc-OSu), to afford the fully protected amino acid 20 in very high overall yields, after chromatography.
(
1
13
H- and C-NMR spectroscopy showed all proton and carbon resonances as expected for the structure
of 20. At this point, we decided to check whether the chirality of the asymmetric carbon is preserved
after the oxidation and unblocking/protection steps. Polarimetric measurements performed on a
sample of pure 20 furnished an optical rotation similar to that obtained for a commercial specimen of
the same compound, at the same concentration. The methyl ester 21 was quantitatively obtained by
treating 20 with a slight excess of diazomethane in dichloromethane. NMR spectroscopy confirmed
the molecular structure of 21, and its optical rotation was almost equal to that reported in the literature
for the same optically pure compound prepared by a different procedure [51], within experimental
error. This finding unambiguously proved that racemization does not occur in our synthesis of l-Dap,
preserving the chirality of 1 throughout the applied multistep procedure.

Molecules 2020, 25, 1313
10 of 21
3
.7. Couplings of l-Dap Methyl Ester 21
As the conclusive point of our study, the l-Dap methyl ester 21 was subjected to couplings with
both enantiomerically pure l and d optical isomers of Fmoc-Ala-OH, to obtain dipeptides 23 and 24
Scheme 6).
(
Scheme 6. Side-chain elongation of 21: preparation of dipeptides 23 and 24. Experimental conditions:
Step 1 (Fmoc removal), 20% DEA in DCM, 30 min, r.t.; Step 2 (coupling): Fmoc-l-Ala-OH or
Fmoc-d-Ala-OH, HOBt monohydrate/EDC/DIEA/DCM, overnight, r.t.
This experiment was performed in order to verify the possible use of 21 in solution-phase
Fmoc-chemistry-based peptide synthesis, and to check whether racemization of 21 occurs during the
coupling process. Diastereomers 23 and 24 were prepared via a classical solution-phase strategy [78].
Accordingly, 21 was treated with DEA to unblock the 3-NH group, and then separately coupled
2
to both N-Fmoc-alanine enantiomers. The system 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride/1-hydroxybenzotriazole (EDC/HOBt) was adopted to activate the carboxyl function
of both protected alanine enantiomers, in the presence of the Hunig’s base (diisopropylethylamine,
DIEA). Reactions gave dipeptides 23 and 24, which were directly analyzed by RP U-HPLC/MS (see
Supplementary Materials), without pre-injection purification. Instrumental analysis highlighted that
only one product was obtained from each coupling, confirming that the experimental conditions
adopted for the side-chain elongation of 21 proceeded without detectable changes of its chirality.
4
. Materials and Methods
Chemicals and solvents were reagent and HPLC grade (Sigma-Aldrich, Milano, Italy); amino
acids were purchased from Senn Chemicals (Bülack, Switzerland). All reagents were used without
further purification. Solvents (Sigma-Aldrich, Milano, Italy) were purified according to standard
◦
procedures, and freshly distilled under a dry nitrogen current, prior to use. Petroleum ether 60–80 C
was used for TLC and column chromatography. Melting points were determined by a Kofler hot-stage
apparatus (Reichert-Thermovar, Germany), at atmospheric pressure, and they are uncorrected. Optical
3
rotations were measured in a 1-dm (2-mL volume) tube, in methanol (c 1.0), and [
α
] values were
D
◦
◦
determined at 20 C, using a digital polarimeter. All NMR spectra were recorded at 25
-mm tubes (seven inches in length), on an Avance 300 Ultrashielded Spectrometer (Bruker Biospin,
Milano, Italy), equipped with a BB0 probe and a temperature control unit. One-dimensional H- and
C-NMR spectra were recorded at 300.132 MHz and 75.03 MHz, respectively. Deuterated chloroform
for spectroscopy (CDCl , 99.5% isotopically pure, containing 0.1% tetramethylsilane (TMS)) was used
±
0.1 C, using
5
1
13
3
as the solvent for all spectral analysis. Chemical shifts (δ) are reported in parts per million (ppm),
and they are referred to the resonance of the residual proton of the solvent (singlet at 7.25 ppm) and to
the central line (77.0 ppm) of the solvent triplet, in proton and carbon spectra, respectively. Proton
1
13
coupling constants (J) are reported in Hz. Relaxation times (T ) for all H and C carbon nuclei
1
were calculated experimentally for all synthesized compounds, using a pulse sequence and method
as published in the literature [79] and fixed to the superior limits of 3 s (proton) and 10 s (carbon).
Extensive proton decoupling experiments were applied to unequivocally assign all resonances to the
1
1
respective protons. Two-dimensional homonuclear correlation spectra ( H, H-TOCSY) were obtained
by the M.Levitt’s composed pulse decoupling sequence (MLEV) for isotropic conditions, with an 80 ms

Molecules 2020, 25, 1313
11 of 21
spin-lock period, elaborating spectral data as previously reported [80]. Acquisition and elaboration
1
13
parameters for one-dimensional H and C spectra were as already published [81]. Reverse-phase
ultrafast HPLC/mass spectrometry (RP U-HPLC/MS) was carried out using a Vantage RP U-HPLC
instrument (Thermo), coupled to a triple quadrupole mass spectrometer and equipped with a heated
electrospray ion source (HESI II), which operated in positive mode [82]. Chromatographic runs were
obtained on a C-18 RP analytical column (Hypersyl Gold, 2.1
×
50 mm, particle size 1.9 µm), under
isocratic conditions and with a 0.4 mL/min flux, applying a 55:45 water (containing 0.1% TFA)/methanol
elution gradient. ESI(+) MS spectra were obtained by a Q-star i-pulsar instrument (AB Sciex), operated
in positive mode at 20 keV, and equipped with a TOF ion analyzer [82]. HRMS analyses were performed
using a MALDI-TOF/TOF 5800 spectrometer (AB Sciex), in reflectron positive mode [83
were monitored by thin-layer chromatography (TLC), using pre-coated silica gel glass plates (Merck,
0-F254, 0.25 mm). The best elution of carboxylic acids was obtained by adding glacial acetic acid (three
,84]. Reactions
6
drops) to the eluent mixture (5 mL). Chromatographic spots were visualized with an ultraviolet (UV)
lamp (254 nm), ninhydrine (0.2% in n-butanol), vanilline solution (5% in ethanol/H SO ), and H SO
4
2
4
2
◦
(
20% in absolute ethanol). After elution, charring at 200 C was applied to develop spot colorations.
Kieselgel 60H without gypsum (Merck) was used as the stationary phase for short-column flash
chromatography, adopting the same gradients used for the respective TLC analyses, with the polar
component being reduced four-fold. All reactions were carried out with pre-flamed glassware and
under an inert atmosphere (dry N ). Diazomethane in DCM (0.66 M solution) was freshly prepared
2
starting from N-methyl-N-nitrosourea, accordingly to the published procedure [74], and then stored
◦
on KOH pellets at
−
20 C. Diazomethane concentration was obtained by back-titration with a standard
solution of benzoic acid.
4
.1. Synthesis of the Weinreb–Nahm Amide 2
HOBt monohydrate (239.6 mg, 1.57 mmol), DIC (0.24 mL, 1.57 mmol), and DIEA (0.27 mL,
α
1
.57 mmol) were added to a solution of N -Fmoc-O-tert-butyl-d-serine (
1; 500 mg, 1.30 mmol)
in dry DCM (5 mL), under magnetic stirring. After 2 h at room temperature, a solution of
N,O-dimethylhydroxylamine hydrochloride (127.2 mg, 1.304 mmol) and DIEA (1.2 equivalents,
0.27 mL, 1.57 mmol) in dry DCM (5 mL) was added dropwise, and the resulting mixture was
maintained under magnetic stirring overnight at room temperature (TLC: diethyl ether/petroleum
ether, 80:20 v/v). The solvent was removed under reduced pressure condition, and the recovered solid
residue was re-dissolved in 5% aqueous NaHSO (10 mL), then extracted with diethyl ether (3
×
10 mL).
4
The organic layers were washed with 5% aqueous NaHCO (3 × 10 mL) and brine (3 × 10 mL), dried
3
over Na SO , paper filtered, and evaporated to dryness under reduced pressure conditions. The crude
2
4
product was purified by flash column chromatography (FCC) eluent: diethyl ether/petroleum ether,
◦
1
9
5:5 v/v), to obtain the title product. White solid (522.7 mg, 94%), mp 136–138 C; H-NMR (300 MHz,
CDCl3) 7.75 (d, 2H, J = 7.0, ArH Fmoc), 7.61 (d, 2H, J = 7.0, ArH Fmoc), 7.38 (t, 2H, J = 7.0, ArH Fmoc),
.30 (t, 2H, J = 7.0, ArH Fmoc), 5.86 (d, 1H, J = 7.2, NH), 4.88 (bs, 1H, CHNH), 4.36 (d, 2H, J = 6.9,
δ
7
13
CH Fmoc), 4.23 (t, 1H, J = 6.9, CH Fmoc), 1.16 (s, 9H, CH tBu); C-NMR (75 MHz, CDCl3)
δ
170.6,
2
3
1
55.9, 143.7, 141.1, 127.5, 126.9, 115.0, 119.7, 73.4, 66.9, 61.8, 61.3, 51.6, 46.9, 32.0, 27.1; MALDI HRMS:
+
calculated 427.2227 for C H N O , found 427.2244.
24
31
2
5
4
.2. Synthesis of the Aldehyde 3
A solution of (522,7 mg, 1.23 mmol) in dry THF was treated with LiAlH (93.1 mg, 2.45 mmol),
under magnetic stirring at room temperature. Reaction was complete in 12 min, as checked by TLC
diethyl ether/petroleum ether 70:30, v/v). A 5% aqueous solution of NaHCO was added (CAUTION),
2
4
(
3
and the mixture was stirred for 10 min. The ethereal phase was separated, and the aqueous residue
was washed with fresh diethyl ether (3
a 5% aqueous solution of NaHSO (3
paper-filtered, and evaporated to dryness under reduced pressure conditions, to give 3. The aldehyde
×
10 mL). The organic extracts were collected, washed with
×
10 mL) and once with brine (10 mL), dried over Na SO ,
4
2
4

Molecules 2020, 25, 1313
12 of 21
was pure enough and was used in the next step without further purification. Colorless oil (415.3 mg,
1
9
2%); TLC (diethyl ether/petroleum ether 70:30, v/v): R = 0.65; H-NMR (300 MHz, CDCl )
δ
9.63
f
3
(d, 1H, J = 2.8, CHO), 7.75 (d, 2H, J = 7.0, ArH), 7.62 (d, 2H, J = 7.0, ArH), 7.40 (t, 2H, J = 7.0, ArH),
7
.32 (t, 2H, J = 7.0, ArH), 5.71 (d, 1H, J = 6.0, NH), 4.42 (d, 2H, J = 7.1, CH Fmoc) 4.32-4.38 (m, 1H,
2
CHNH), 4.25 (t, 1H, J = 7.1, CH Fmoc), 3.96 (dd, 1H, J = 11.2 and 7.6, CH O), 3.63 (dd, 1H, J = 11.2
2
13
and 5.8, CH O), 1.16 (s, 9H, CH ); C-NMR (75 MHz, CDCl3)
1
δ
199.1, 156.1, 143.7, 141.2, 127.7, 127.0,
2
3
+
25.0, 119.9, 73.7, 67.1, 60.4, 59.9, 47.0, 27.1; MALDI HRMS: calculated for C H NO 368.1858, found
2
2
26
4
3
68.1869.
4
.3. Synthesis of 4–10. General Procedure for the Reductive Amination
To a solution of
3 (206.6 mg, 0.56 mmol) in dry ethanol (4 mL), Ti(OiPr) (0.34 mL, 1.12 mmol)
4
was added under magnetic stirring at room temperature. After 10 min, the appropriate primary
amine or arylsulfonylamide (0.67 mmol) was added. The resulting mixture was maintained under
stirring at room temperature until complete conversion of 3, monitoring the imine formation by
TLC (diethyl ether/petroleum ether, 80:20 v/v), and NaBH CN (52.8 mg, 0.84 mmol) was added.
3
The mixture was stirred overnight at room temperature, and brine (5 mL) was then added. The white
precipitate was filtered off by paper filtration and washed with a minimum amount of absolute ethanol.
The mother liquor was concentrated under reduced pressure conditions, and the residue was eluted
®
with ethyl acetate through a short pad of Celite 545 . The organic solution was dried over MgSO ,
4
paper-filtered, and evaporated to dryness under vacuum, to give a crude material that was fractioned by
column chromatography (eluent: AcOEt/n-hexane, 50:50 v/v, reducing the polar component four-fold),
to deliver the desired 2,3-diaminopropanols 4–10 in an 82%–92% overall yield.
2
0
7
,3-Diaminopropanol
.17; H-NMR (300 MHz, CDCl )
.28–7.43 (m, 9H, ArH Fmoc and ArH phenyl), 5.52 (d, 1H, J = 6.8, NHCO), 4.37 (d, 2H, J = 6.9, CH2
4
. Colorless oil (236.9 mg, 92%); TLC (diethyl ether/n-hexane 80:20, v/v): R =
f
1
δ 7.76 (d, 2H, J = 7.0, ArH Fmoc), 7.60 (d, 2H, J = 7.0, ArH Fmoc),
3
Fmoc), 4.22 (1H, J = 6.9, CH Fmoc), 3.77–3.92 (m, 4H, CH, CH O and PhCH ), 3.41–3.49 (m, 1H, CH O),
2
2
2
1
3
2
.73–2.81 (m, 2H, CH N), 2.25–2.40 (m, 1H, NH), 1.19 (s, 9H, CH tBu); C-NMR (75 MHz, CDCl3)
2
3
δ
156.6, 144.1, 141.2, 136.0, 128.7, 127.93, 127.91, 126.6, 124.7, 124.2, 119.8, 73.7, 68.5, 66.8, 55.4, 52.8,
+
+
48.0, 47.1, 27.1; ESI(+) MS: m/z 459.38 [M + H] ; MALDI HRMS: calculated for C H N O 459.2642,
29 35 2 3
found 459.2658.
2
0
,3-Diaminopropanol 5. Pale yellow oil (280.2 mg, 90%); TLC (diethyl ether/n-hexane 80:20, v/v): R =
f
1
.25; H-NMR (300 MHz, CDCl ) severe overlap, 75:25 mixture of two conformers,
δ
7.79 (d, 4H, J = 6.9,
3
ArH Fmoc), 7.65 (d, 4H, J = 6.9, ArH Fmoc), 7.19–7.49 (m, 28H, ArH Fmoc and ArH phenyls), 5.76 (d,
.25H, J = 8.9, NHCO minor conformer), 5.55 (d, 0.75H, J = 7.2, NHCO major conformer), 4.86 (sb, 2H,
CHPh ), 4.44–4.48 (m, 0.25H, CHNH minor conformer), 4.42 (d, 4H, J = 8.0, CH Fmoc), 4.22 (t, 2H, J
0
2
2
=
8.0, CH Fmoc), 4.09–4.16 (m, 0.75H, CHNH major conformer), 3.81–3.86 (m, 0.50H, CH O minor
2
conformer), 3.45–3.69 (m, 1.5H, CH O major conformer), 2.70–2.80 (m, 2H, CH N major conformer),
2
2
13
2
.12–2.39 (m, 4H, CH N minor conformer, and NH both conformers), 1.17 (s, 18H, CH tBu); C-NMR
2 3
(
75 MHz, CDCl3)
δ
156.1, 155.8, 145.3, 144.1, 143.9, 143.7, 143.6, 143.0, 141.2, 128.7, 128.6, 128.4, 127.7,
1
5
27.6, 127.5, 127.2, 127.1, 126.90, 126.88, 126.81, 125.0, 124.7, 120.0, 119.9, 73.8, 72.9, 67.2, 67.0, 66.6,
+
0.8, 50.5, 49.1, 47.2, 47.0, 27.3, 27.2; ESI(+) MS: m/z 557.72 [M + H] ; MALDI HRMS: calculated for
+
C H N NaO 557.2775, found 557.2797.
39
38
2
3
2
0
,3-Diaminopropanol
.21; H-NMR (300 MHz, CDCl )
6
. Colorless oil (237.9 mg, 90%); TLC (diethyl ether/n-hexane 80:20, v/v): R =
f
1
δ
7.76 (d, 2H, J = 7.2 Hz, ArH Fmoc), 7.61 (d, 2H, J = 7.2 Hz, ArH
3
Fmoc), 7.40 (t, 2H, J = 7.2 Hz, ArH Fmoc), 7.20–7.40 (m, 7H, ArH Fmoc and ArH phenyl), 5.43 (d, 1H,
J = 7.9, NHCO), 4.38-4.48 (m, 1H, CH Fmoc), 4.21–4.36 (m, 2H, CH Fmoc), 3.68–3.82 (m, 2H, CH O
2
2
and CH CHNH), 3.54–3.97 (m, 1H, CHNH), 2.60–2.76 (m, 1H, CH NH), 2.23–2.46 (m, 2H, CH NH and
2
2
2
13
CHNH), 1.34 (d, 3H, J = 6.5, CH ), 1.14 (s, 9H, CH tBu); C-NMR (75 MHz, CDCl3)
δ
156.3, 145.8,
3
3

Molecules 2020, 25, 1313
13 of 21
1
43.4, 141.3, 129.4, 128.4, 127.6, 127.5, 127.0, 126.5, 125.0, 119.9, 73.2, 66.9, 62.2, 55.2, 47.4, 42.1, 27.5, 23.4;
+
+
ESI(+) MS: m/z 473.42 [M + H] ; MALDI HRMS: calculated for C H N O 473.2799, found 473.2817.
30
37
2
3
2
0
7
,3-Diaminopropanol
.21; H-NMR (300 MHz, CDCl )
.40 (t, 2H, J = 7.3, Ar Fmoc), 7.19–7.39 (m, 7H, ArH Fmoc and ArH phenyl), 5.52 (d, 1H, J = 7.8,
7
. Colorless oil (240.5 mg, 91%); TLC (diethyl ether/n-hexane 80:20, v/v): R =
f
1
δ 7.77 (d, 2H, J = 7.3, ArH Fmoc), 7.60 (d, 2H, J = 7.3, ArH Fmoc),
3
NHCO), 4.46–4.21 (m, 3H, CH and CH Fmoc), 3.63–3.92 (m, 2H, CH O), 3.55–3.62 (m, 1H, CHCH N
2
2
2
and CHNH), 2.50–2.72 (m, 3H, CH N and CHNH), 1.16 (d, 3H, J = 6.8, CH ), 1,13 (s, 9H, CH tBu);
2
3
3
1
3
C-NMR (75 MHz, CDCl3)
δ
157.1, 145.8, 144.0, 141.3, 129.4, 128.7, 127.6, 127.5, 126.4, 126.4, 125.3, 115.0,
3.2, 63.0, 53.5, 50.5, 48.8, 47.3, 27.3, 24.2; ESI(+) MS: m/z 473.35 [M + H] ; MALDI HRMS: calculated
for C H N O 473.2799, found 473.2780.
+
7
+
30
37
2
3
2
,3-Diaminopropanol 8. Colorless oil (204.3 mg, 89%); TLC (diethyl ether/n-hexane 80:20, v/v): R = 0.14;
f
1
H-NMR (300 MHz, CDCl3)
δ 7.65–7.80 (m, 2H, ArH), 7.61 (d, 2H, J = 7.0, ArH), 7.26-7.45 (m, 4H, ArH),
5
.63 (bs, 1H, NHCO), 4.38 (d, 2H, J = 6.9, CH Fmoc), 4.23 (1H, J = 6.9, CH Fmoc), 3.31–3.45 (m, 3H,
2
CH and CH O), 2.51–2.90 (m, 4H, CH N), 1.30-1.58 (m, 2H, CH CH ), 1.16 (s, 9H, CH tBu), 0.75–0.90
2
2
2
3
3
13
(
7
m, 3H, CH CH ); C-NMR (75 MHz, CDCl3) δ 156.2, 143.7, 141.3, 127.8, 127.2, 125.0, 124.8, 119.9,
3 2
+
5.6, 67.5, 52.0, 48.3, 27.2, 21.6, 11.9; ESI(+) MS: m/z 411.29 [M + H] ; MALDI HRMS: calculated for
+
C H N O 411.2642, found 411.2660.
25
35
2
3
2
=
,3-Diaminopropanol
0.24; mp 127–129 C; H-NMR (300 MHz, CDCl )
9
. Pale yellow solid (253.9 mg, 82%); TLC (diethyl ether/n-hexane 80:20, v/v): R
f
◦
1
δ
8.20–8.34 (m, 2H, SO ArH), 7.90–8.10 (m, 2H,
3
2
SO ArH), 7.76 (d, 2H, J = 7.2, ArH Fmoc), 7.55 (d, 2H, J = 7.2, ArH Fmoc), 7.33–7.44 (m, 2H, ArH Fmoc),
2
7
.20–7.32 (m, 2H, ArH Fmoc), 5.88 (bs, 1H, NHSO ), 5.32 (d, 1H, J = 7.6, NHCO), 4.35–4.49 (m, 1H,
2
CHNH), 4.29–4.35 (m, 2H, CH Fmoc), 4.10–4.19 (m, 1H, CH Fmoc), 4.00–4.09 (m, 1H, CH O), 3.73–3.87
2
2
13
(
(
5
5
m, 1H, CH O), 3.15–3.31 (m, 1H, CH N), 3.00–3.15 (m, 1H, CH N), 1.16 (s, 9H, CH tBu); C-NMR
2
2
2
3
75 MHz, CDCl3)
δ
156.4, 150.0, 145.9, 143.7, 141.3, 128.2, 127.8, 127.0, 124.9, 124.3, 120.0, 73.9, 67.1, 62.3,
+
+
0.1, 47.1, 42.1, 27.3; ESI(+) MS: m/z 554.38 [M + H] ; MALDI HRMS: calculated for C H N O S
28 32 3 7
54.1955, found 554.1938.
2
0
,3-Diaminopropanol 10. White solid (239.7 mg, 85%); TLC (diethyl ether/n-hexane 80:20, v/v): R =
f
◦
.22; mp 119–121 C; H-NMR (300 MHz, CDCl ) δ 7.68-7.80 (m, 4H, SO ArH), 7.58 (d, 2H, J = 7.4,
3 2
1
ArH Fmoc), 7.40 (t, 2H, J = 7.4, ArH Fmoc), 7.23–7.35 (m, 4H, SO ArH and ArH Fmoc), 5.32–5.48 (m,
2
2
H, NHCO and NHSO ), 4.33 (d, 2H, J = 7.2, CH Fmoc), 4.19 (t, 1H, J = 7.2, CH Fmoc), 3.75–3.85 (m,
2 2
1
H, CHNH), 3.40–3.50 (m, 2H, CH O), 3.16-3.28 (m, 1H, CH N), 3.00–3.11 (m, 1H, CH N), 2.37 (s, 3H,
2
2
2
1
3
CH tosyl), 1.15 (s, 9H, CH tBu); C-NMR (75 MHz, CDCl3)
δ
156.2, 141.2, 136.7, 129.7, 127.8, 127.5,
3
3
+
1
27.0, 125.1, 124.7, 120.0, 73.7, 66.9, 65.0, 50.3, 49.7, 47.1, 45.6, 27.3, 21.4; ESI(+) MS: m/z 523.65 [M + H] ;
+
MALDI HRMS: calculated for C H N O S 523.2261, found 523.2287.
29
35
2
5
4
.4. Methylation of 9. Synthesis of 2,3-diaminopropanol 11
Compound (100.0 mg, 0.18 mmol) was methylated with trimethyloxonium tetrafluoroborate
66.4 mg, 0.45 mmol), in the presence of DIEA (0.11 mL, 0.63 mmol), in DCM (20 mL), accordingly
9
(
to the previously published procedure [76]. Pale yellow oil (102.1 mg, quantitative); TLC (diethyl
1
ether/n-hexane 80:20 v/v) R = 0.48; H-NMR (300 MHz, CDCl )
δ 8.29–8.44 (m, 2H, SO ArH), 7.91–8.09
2
f
3
(
m, 2H, SO ArH), 7.70–7.80 (m, 2H, ArH Fmoc), 7.50-7.65 (m, 2H, ArH Fmoc), 7.31–7.42 (m, 2H, ArH
2
Fmoc), 7.23–7.31 (m, 2H, ArH Fmoc), 5.34 (d, 1H, J = 7.1, CHNH), 4.30–4.41 (m, 2H, CH Fmoc),
2
4
.12-4.28 (m, 1H, CH Fmoc), 3.75–3.97 (m, 1H, CHNH), 3.60–3.73 (m, 1H, CH O), 3.32–3.48 (m, 2H,
2
13
CH O and CH N), 3.10-3.21 (m, 1H, CH N), 2.83 (s, 3H, CH ), 1.16 (s, 9H, CH tBu); C-NMR (75 MHz,
2
2
2
3
3
CDCl3)
δ 156.1, 150.0, 143.6, 141.2, 127.7, 127.0, 125.0, 124.7, 124.4, 120.0, 73.5, 66.9, 60.5, 54.7, 51.0, 49.1,
+
+
4
7.1, 35.7, 27.2; ESI(+) MS: m/z 568.19 [M + H] ; MALDI HRMS: calculated for C H N O S 568.2112,
29 34 3 7
found 568.2133.

Molecules 2020, 25, 1313
14 of 21
4
.5. Synthesis of 2,3-diaminopropanol 12
To a solution of 10 (100.0 mg, 0.19 mmol), in dry DCM (3 mL), TFA (1 mL) and TFE (0.3 mL)
were added. The reaction mixture was magnetically stirred at room temperature for 15 min (TLC:
diethyl ether/petroleum ether 80:20, v/v). After complete conversion of 10, the mixture was evaporated
to dryness under reduced pressure conditions. The residue was dissolved in DCM (5 mL), treated
with solid Na CO (CAUTION) until complete gas evolution, and then desalted by elution with small
2
3
volumes of DCM on a short silica gel pad. The organic solution was dried over Na SO , and evaporated
2
4
to dryness under reduced pressure conditions, to afford 12 (88.5 mg, quantitative). White solid,
◦
1
mp 142–145 C; TLC (diethyl ether/n-hexane 80:20, v/v): R = 0.16; H-NMR (300 MHz, CDCl )
δ
f
3
7
7
.62–7.80 (m, 4H, ArH Fmoc and ArH Ts), 7.45–7.60 (m, 2H, ArH Fmoc), 7.30–7.40 (m, 2H, ArH Fmoc),
.19–7.30 (m, 4H, ArH Fmoc and ArH Ts), 5.52–5.73 (m, 1H, NHCO), 5.18 (bs, 1H, NHSO ), 4.00–4.44
2
(
m, 4H, CH Fmoc, CH Fmoc and CHNH), 3.52–3.78 (m, 2H, CH O), 3.01 (bs, 1H, OH), 2.50–2.71
2
2
13
(m, 2H, CH NH), 2.32 (s, 3H, CH ); C-NMR (75 MHz, CDCl3)
δ
156.6, 143.5, 141.2, 139.0, 136.4,
2
3
1
29.8, 127.7, 123.1, 126.3, 125.1, 119.9, 67.0, 62.9, 53.4, 47.0, 43.2, 21.4; MALDI HRMS: calculated for
+
C H N O S 467.1635, found 467.1659.
25
27
2
5
4
.6. Preparation of Fmoc-l-Dap(Ts)-OCH (14)
3
Compound 12 (232.8 mg, 0.5 mmol) was dissolved in acetone (5 mL), and aqueous 15% solution of
NaHCO (3 mL) was added, followed by solid NaBr (10.2 mg, 0.1 mmol). TEMPO (7.5 mg, 0.05 mmol)
3
◦
was added to the mixture, under magnetic stirring at 0 C. After slow addition of TCCA (220.5 mg,
.0 mmol) within 20 min at 0 C, the mixture was maintained under magnetic stirring overnight at
◦
5
room temperature. Then, 2-propanol (3 mL) was added, and the mixture was filtered on a short pad of
®
Celite 545 , concentrated under vacuum, and treated with 5 mL of a saturated aqueous solution of
Na CO . The aqueous phase was washed with small portions of AcOEt (3 × 10 mL), treated with 5%
2
3
aqueous KHSO (10 mL), and re-extracted with AcOEt (3
×
10 mL). The organic layers were dried
4
over MgSO and paper-filtered, and the solvent was evaporated to dryness, yielding a raw material
4
which was directly treated with an excess of diazomethane (0.66 N solution in dry DCM, 4 mL), under
magnetic stirring for 15 min at room temperature [85]. The mixture was evaporated to dryness under
reduced pressure conditions (CAUTION: use a fume hood), to give 14. White solid (227.2 mg; 92%,
◦
over 2 steps; 68% total yield, based on the initial amount of the amino acid
1
, 1.30 mmol); mp >90 C,
1
dec.; TLC (diethyl ether/n-hexane 80:20, v/v) R = 0.36; H-NMR (300 MHz, CDCl )
δ 7.65–7.79 (m, 4H,
f
3
Ar Fmoc and ArH Ts), 7.52–7.61 (m, 2H, ArH Fmoc), 7.35–7.43 (m, 2H, ArH Fmoc), 7.22–7.34 (m, 5H,
Ar Fmoc, ArH Ts and NHSO ), 5.89 (d, 1H, J = 6.5 Hz, NHCO), 4.48-4.62 (m, 4H, CH Fmoc, CH Fmoc
2
2
and CHNH), 3.95 (dt, 1H, J = 21 and 11 Hz, CH N), 3.94 (dt, 1H, J = 21 and 11 Hz, CH N), 3.77 (s,
2
2
13
3
H, OCH ), 2.38 (s, 3H, CH ); C-NMR (75 MHz, CDCl3)
δ
171.1, 156.8, 143.6, 141.3, 123.7, 129.6,
3
3
1
28.6, 127.7, 127.0, 126.4, 125.0, 119.9, 67.1, 56.0, 52.7, 47.1, 41.8, 22.1; MALDI HRMS: calculated for
+
C H N O S 495.1584, found 495.1606.
26
27
2
6
4
.7. Synthesis of Boc-l-Dap(Fmoc)-OH (20): Multistep Procedure
Step 1: Acidolysis. To a solution of (229 mg, 0.5 mmol), in dry DCM (3 mL), TFA (2 mL) and
TFE (0.3 mL) were added. The reaction mixture was magnetically stirred at room temperature for
5 min (TLC: diethyl ether/petroleum ether 80:20, v/v). After complete conversion of , the mixture was
4
1
4
evaporated to dryness under reduced pressure conditions. The residue was dissolved in DCM (5 mL),
®
treated with solid Na CO until complete gas evolution, and desalted on a short pad of Celite 545 by
2
3
elution with small volumes of DCM. The organic phase was dried over Na SO and evaporated to
2
4
dryness under reduced pressure conditions, to afford a crude solid which was directly used in the
next step, without purification. Step 2: Oxidation. The crude material obtained from the previous
step was subjected to oxidation, applying the procedure above described for the preparation of the
methyl ester 14. The final crude product was dried under vacuum, and then subjected to the next

Molecules 2020, 25, 1313
15 of 21
step without further purification. Step 3: Removal of Fmoc group. The crude product recovered
from step 2, was reacted with 5 mL of a 20% DEA solution in DCM, under magnetic stirring at room
temperature for 30 min. Evaporation to dryness of volatile gave a solid residue that was dissolved in
AcOEt and precipitated with cold n-pentane. The solid was filtered, washed three times with small
portions of n-pentane, and dried under vacuum. The crude material, as recovered, was immediately
subjected to the next step of the procedure. Step 4: Protection with Boc group. The crude solid
recovered from step 3 was suspended in H O (5 mL), containing NaHCO (two equivalents with
2
3
respect to
4; 88 mg, 1 mmol), and a solution of di-tert-butyl-dicarbonate (1.1 equivalents with respect to
4
; 120 mg, 0.55 mmol) in 1,4-dioxane (5 mL) was added dropwise to the reaction system. The resulting
clear mixture was maintained under stirring overnight at room temperature, and then concentrated
under reduced pressure conditions to give an aqueous residue which was made acidic by adding
5
% aqueous KHSO , before being extracted with ethyl acetate (3
×
10 mL). The organic layers were
4
collected, washed once with brine (10 mL), dried over Na SO , paper-filtered, and evaporated to
2
4
dryness to give a white solid which was transformed in a white powder by co-evaporation from a
:1 (v/v) methyl-tert-butyl ether (MTB)/n-hexane mixture. TLC (CHCl /MeOH 90:10, v/v): R = 0.34.
2
3
f
Step 5: Hydrogenolysis of benzyl group. The crude product recovered from step 4 was dissolved in
◦
absolute ethanol (5 mL), and the reaction flask was immersed in a water bath at 25 C. To the alcoholic
solution, 10% palladium-carbon (22.9 mg) and 1,4-cyclohexadiene (10 equivalents with respect to
4
; 0.47 mL, 5.0 mmol) were added, and the mixture was allowed to react under magnetic stirring
®
overnight. After filtration over a short pad of Celite 545 , the solvent was evaporated under reduced
pressure conditions, and the solid residue was partitioned in a 1:1 (v/v) H O/EtOAc mixture (10 mL).
2
The aqueous phase was separated, back extracted with EtOAc (3
pressure conditions, and lyophilized. Step 6: Protection with Fmoc group. The crude product obtained
×
10 mL), concentrated under reduced
from step 5 was dissolved in a 1:1 (v/v) 1,4-dioxane/H O mixture (15 mL) containing NaHCO (two
2
3
equivalents with respect to
to ; 185.5 mg, 25 mmol) in 1,4-dioxane (5 mL) was added dropwise in 35 min, and the resulting
mixture was magnetically stirred overnight at room temperature. The reaction was checked by TLC
CHCl /MeOH 95:5, v/v; ninhydrin as the visualizing agent), and, after complete disappearing of the
4; 88 mg, 1 mmol). A solution of Fmoc-OSu (1.1 equivalents with respect
4
(
3
free amino compound, the solution was concentrated under vacuum, and the aqueous residue was
made acidic (pH = 2.5) by adding 5% aqueous KHSO and extracted with ethyl acetate (3 10 mL).
The collected organic layers were washed once with brine (10 mL), dried over Na SO , paper=filtered,
×
4
2
4
and evaporated to dryness to give a viscous residue which was rapidly dissolved in the smallest
volume of DCM and precipitated from cold n-pentane. The precipitate was filtered under vacuum
and dried by using an oil pump. The final product was purified by column chromatography. White
◦
powder (404.7 mg; 93% over six steps; 73% based on the initial amount of
1
, 1.30 mmol); mp >78 C
20
1
(
dec.); TLC (CHCl /MeOH 90:10, v/v): R = 0.35; [α]
=
−
11.8 (c 1.0, CH OH); H-NMR (300 MHz,
δ 7.74 (d, 2H, J = 6.2 Hz, ArH Fmoc), 7.56 (d, 2H, J = 6.2 Hz, ArH Fmoc), 7.36 (t, 2H, J = 6.2 Hz,
3
f
D
3
CDCl3)
ArH Fmoc), 7.29 (t, 2H, J = 6.2 Hz, ArH Fmoc), 5.71 (bs, 1H, NHCO), 5.40 (bs, 1H, NHCO), 4.41 (d, 2H,
J = 6.1 Hz, CH Fmoc), 4.24–4.36 (m, 1H, CHNH), 4.19 (t, 1H, J = 6.1 Hz), 3.51–3.72 (m, 2H, CH NH),
2
2
13
1
6
.45 (s, 9H, tBu); C-NMR (75 MHz, CDCl3)
7.5, 54.8, 47.2, 42.7, 34.0, 28.1. MALDI HRMS: calculated for C H N O 427.1864, found 427.1881.
δ
172.3, 157.6, 157.1, 143.8, 141.4, 127.8, 127.1, 125.0, 120.0,
+
23
27
2
6
4
.8. Methylation of 20. Synthesis of Fmoc-L-Dap(Boc)-OCH (21)
3
The purified amino acid 20 (404.7 mg, 0.95 mmol) was treated with an excess of a 0.66 N solution
of diazomethane in dry DCM (5 mL), under magnetic stirring, for 15 min at room temperature.
The mixture was evaporated to dryness under reduced pressure conditions (CAUTION: use a fume
hood), and the solid residue was co-evaporated twice from small portions of a 2:1 (v/v) MTB/n-pentane
mixture, to give pure 21 without need for chromatography. White powder (417.8 mg, quantitative);
◦
20
mp >83 C (dec.); TLC (CHCl /MeOH 90:10, v/v): R = 0.65. [α]
=
−
12.5 (c 1.0, CH OH; Lit. [51]:
−
10
3
f
D
3
1
and 14; H-NMR (300 MHz, CDCl ) δ 7.76 (d, 2H, J = 6.2 Hz, ArH Fmoc), 7.57 (d, 2H, J = 6.2 Hz,
−
3

Molecules 2020, 25, 1313
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ArH Fmoc), 7.39 (t, 2H, J = 6.2 Hz, ArH Fmoc), 7.28 (t, 2H, J = 6.2 Hz, ArH Fmoc), 5.32 (bs, 1H, NHCO),
5
.11 (bs, 1H, NHCO), 5.40 (bs, 1H, NHCO), 4.30–4.42 (m, 3H, CHNH and CH Fmoc), 4.21 (t, 1H, J =
2
13
6
.1 Hz, CH Fmoc), 3.75 (s, 3H, OCH ), 3.50-3.63 (m, 2H, CH NH), 1.45 (s, 9H, tBu); C-NMR (75 MHz,
3
2
CDCl ) δ 171.0, 156.7, 155.4, 143.9, 141.4, 127.7, 127.1, 125.0, 120.0, 80.4, 67.1, 54.2, 52.6, 47.3, 43.2, 28.3;
3
+
MALDI HRMS: calculated for C H N O 441.2020, found 441.1998.
24
29
2
6
4
.9. Coupling of 21 with Fmoc-l-Ala-OH and Fmoc-d-Ala-OH. Synthesis of diastereomers 23 and 24
DEA (2 mL) was added to a solution of 21 (356.4 mg, 0.81 mmol), in dry DCM (5 mL), under
magnetic stirring at room temperature. After 30 min, the volatile was removed under reduced pressure
conditions, to afford a solid residue which was dissolved in the minimum volume of EtOAc and
precipitated from cold n-pentane. The supernatant was separated, and the solid residue was washed
with small portions of cold n-pentane (5
product 22 was dissolved in DCM (3 mL) and added to a magnetically stirred mixture of DIEA (0.35 mL;
.03 mmol), HOBt monohydrate (150 mg; 0.97 mmol), and EDC (160 mg; 0.85 mmol) in DCM (5 mL).
×
5 mL), then dried under vacuum. The recovered crude
2
After 15 min at room temperature, the appropriate enantiomer of Fmoc-Ala-OH (311.3 mg; 1.0 mmol),
dissolved in DCM (3 mL), was added dropwise, and the mixture was allowed to stir overnight at room
temperature. The solvent was then evaporated to dryness under reduced pressure conditions, and the
residue was solubilized in EtOAc (10 mL), washed once with 5% aqueous NaHCO (5 mL), once with
3
5
% aqueous KHSO (5 mL), and once with brine (5 mL). The organic layer was dried over Na SO ,
4
2
4
paper-filtered, and evaporated to dryness to yield the expected dipeptides 23 and 24, both as a white
powder. The pair of crude diastereomers was directly analyzed by RP U-HPLC/MS, without further
pre-injection purification (Supplementary Materials).
5
. Conclusions
In summary, a synthetic route is proposed for the multi-step preparation of orthogonal di-protected
α
l-Dap methyl esters. In the proposed procedure, commercial N -Fmoc-O-tert-butyl-d-serine is initially
transformed into the corresponding serinal. This compound is subjected to a Ti(IV)-assisted reductive
amination with amines and arylsulfonamides, to give 2,3-diaminopropanols which are in turn oxidized
by the TCCA/TEMPO system. The obtained carboxylic acids are finally methylated, to yield the targeted
esters in 68%–73% overall yields. Reductive amination allows also the preparation of N-methylated
and N-ethylated 2,3-diaminopropanol intermediates as the most likely precursors of N-alkylated
l-Dap scaffolds. Chromatographic purifications are minimized through the entire procedure. The total
preservation of chirality enhanced the synthetic utility of the method, which can successfully be used
to prepare protected l-Dap building blocks for a broad application in the synthesis of biologically
active peptides.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/6/1313/s1:
1
13
1
13
Figure S1: H spectrum of
2
; Figure S2: C spectrum of
4
2
; Figure S3: H spectrum of
3
; Figure S4: C spectrum of
1
13
1
13
3
of
; Figure S5: H spectrum of
; Figure S9: H spectrum of
spectrum of
; Figure S6: C spectrum of
4
; Figure S7: H spectrum of
; Figure S11: H spectrum of
; Figure S14: C spectrum of
5
; Figure S8: C spectrum
1
; Figure S10: 13C spectrum of
1
13
5
6
6
7
; Figure S12:
C
1
13
1
7
; Figure S13: H spectrum of
8
8
; Figure S15: H spectrum of
9
; Figure
1
3
1
13
1
S16: C spectrum of
9
; Figure S17: H spectrum of 10; Figure S18: C spectrum of 10; Figure S19: H spectrum of
11
; Figure S20: 13C spectrum of 11; Figure S21: H spectrum of 12; Figure S22: C spectrum of 12; Figure S23: H
1
13
1
spectrum of 13; Figure S24: 13C spectrum of 13; Figure S25: H spectrum of 20; Figure S26: C spectrum of 20
1
13
;
1
13
Figure S27: H spectrum of 21; Figure S28: C spectrum of 21; Figures S29–S36: ESI(+) MS spectra of
S37: RP U-HPLC analysis of crude 23 and crude 24; Figures S38 and S39: ESI(+) MS spectra of 23 and 24.
4–11; Figure
Author Contributions: Conceptualization, C.S., C.M.A., and A.T.; methodology, C.S., A.T., C.M.A., and P.D.L.;
software, C.S.; NMR analysis, C.S.; MS analysis, D.A.; RP U-HPLC/MS analysis, F.M.; polarimetry, P.D.L.;
investigation, C.S.; data curation, C.S., A.T.; writing—original draft preparation, C.S. and A.T.; writing—review
and editing, C.S. and C.M.A.; supervision, C.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.

Molecules 2020, 25, 1313
17 of 21
Acknowledgments: Support from MIUR (Ministero dell’istruzione, dell’Universit
à
e della Ricerca, Italy) is
gratefully acknowledged. This work is dedicated to the memory of Prof. Giovanni Sindona.
Conflicts of Interest: The authors declare no conflict of interest. MIUR (Ministero Italiano dell’Università e della
Ricerca) had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing
of the manuscript, or in the decision to publish the results.
Abbreviations
TEMPO (2:2,6,6-tetramethylpiperidin-1-yl)oxyl, TCCA (trichloroisocyanuric acid); DEA (N,N-diethylamine),
0
DIEA (N,N-diisopropylethylamine); HOBt (1-hydroxybenzotriazole); DIC (N,N -diisopropylcarbodiimide);
EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); Boc O (di-tert-butyl-dicarbonate);
2
Fmoc-OSu (N-(9-fluorenylmethoxycarbonyloxy)succinimide); TFA (trifluoroacetic acid); TFE (trifluoroethanol);
THF (tetrahydrofuran); DCM (dichloromethane); AcOEt (ethyl acetate); CH CN (acetonitrile); MTB (methyl-
3
tert-butyl ether); TLC (thin-layer chromatography); FCC (flash column chromatography); RP U-HPLC/MS
(
reversed-phase ultrafast high-pressure liquid chromatography/mass spectrometry); HRMS (high-resolution mass
spectrometry); ESI(+) MS (positive-mode electro spray ionization mass spectrometry); TOF (time of flight); MS/MS
tandem mass spectrometry); TOCSY (total correlation spectroscopy).
(
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Sample Availability: Samples of compounds are not available from the authors.
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