2,3-Diaminopropanols Obtained from d-Serine as Intermediates in the Synthesis of Protected 2,3-l-Diaminopropanoic Acid (l-Dap) Methyl Esters

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 Nα-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.


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 [1]. 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 [2]. The known preparative methods for the total synthesis of albizziine employs protected derivatives of l-Dap as the starting materials [3]. Bleomycins (BLMs) are structurally related glycopeptide-derived 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 protect the 2-NH2 group of the methyl esters with the base-labile Fmoc group (PG 1 ). In that structure, the 3-NH2 amino function could orthogonally be protected to give a tosyl derivative, or one with an acid-labile Boc group (PG 2 ). In the 2,3-diamminopropanol intermediates, the simultaneous presence of a benzyl moiety and an orthogonal Fmoc protection on the 3-and 2-NH2 functions, respectively, could also support the facile interconversion of the pair of amino protections, enabling the installation of Boc (PG 1 ) and Fmoc (PG 2 ) groups at the 2-and 3-positions of the final L-Dap methyl ester structures.

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 3 with amines and sulfonamides. The protected amino acid 1 reacted with N,O-dimethylhydroxylamine hydrochloride [58] to give the Weinreb-Nahm amide 2, which was recovered in a 94% yield, without need for chromatographic purification. Reduction of 2 by LiAlH4 afforded the corresponding α-amino aldehyde 3, which was obtained in a 92% yield, avoiding chromatography. The structures of compounds 2 and 3 were confirmed by 1 H-and 13 C-NMR spectroscopy. Compound 3 was then subjected to the reductive 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 assisted by the Lewis acid Ti(O i Pr)4, in the presence of sodium cyanoborohydride, a reducing agent which is widely employed in metal-assisted reactions of amino acids and their Scheme 1. The retrosynthetic analysis for the preparation of orthogonally N α ,N β -diprotected 2,3-l-diaminopropanoic acid (l-Dap) methyl esters (PG 1 and PG 2 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 protect the 2-NH 2 group of the methyl esters with the base-labile Fmoc group (PG 1 ). In that structure, the 3-NH 2 amino function could orthogonally be protected to give a tosyl derivative, or one with an acid-labile Boc group (PG 2 ). In the 2,3-diamminopropanol intermediates, the simultaneous presence of a benzyl moiety and an orthogonal Fmoc protection on the 3-and 2-NH 2 functions, respectively, could also support the facile interconversion of the pair of amino protections, enabling the installation of Boc (PG 1 ) and Fmoc (PG 2 ) groups at the 2-and 3-positions of the final l-Dap methyl ester structures.

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 3 with amines and sulfonamides. The protected amino acid 1 reacted with N,O-dimethylhydroxylamine hydrochloride [58] to give the Weinreb-Nahm amide 2, which was recovered in a 94% yield, without need for chromatographic purification. Reduction of 2 by LiAlH 4 afforded the corresponding α-amino aldehyde 3, which was obtained in a 92% yield, avoiding chromatography. The structures of compounds 2 and 3 were confirmed by 1 H-and 13 C-NMR spectroscopy. Compound 3 was then subjected to the reductive 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 assisted by the Lewis acid Ti(O i Pr) 4 , in the presence of sodium cyanoborohydride, a reducing agent which is widely employed in metal-assisted reactions of amino acids and their derivatives [59,60].
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 3 reacted smoothly with benzylamine, in the presence of the titanium(IV) species and the hydride donor, to afford 4 in excellent yields (92%), after chromatography. Thin-layer chromatography (TLC) analysis, as well as the 1 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. The molecular structure of 4 was confirmed by one-and two-dimensional (1D and 2D) NMR spectroscopy. In particular, the 1D 1 H-and 13 C-NMR spectral plots displayed the correct number, multiplicity, and integral values of all resonances assignable to the expected structure of 4. The contour plot of the 2D homonuclear proton TOCSY spectrum of the benzyl derivative 4 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 3.40 ppm, and between 2.60 and 2.90 ppm, which were generated by the resonances of all correlated protons along the molecular backbone of 4 ( Figure 1). Two-dimensional spectroscopy unequivocally 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 2,3diaminopropanols. The molecular structure of 4 was confirmed by one-and two-dimensional (1D and 2D) NMR spectroscopy. In particular, the 1D 1 H-and 13 C-NMR spectral plots displayed the correct number, multiplicity, and integral values of all resonances assignable to the expected structure of 4. The contour plot of the 2D homonuclear proton TOCSY spectrum of the benzyl derivative 4 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 3.40 ppm, and between 2.60 and 2.90 ppm, which were generated by the resonances of all correlated protons along the molecular backbone of 4 ( Figure 1). Two-dimensional spectroscopy unequivocally 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 2,3diaminopropanols. derivatives [59,60]. 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 3 reacted smoothly with benzylamine, in the presence of the titanium(IV) species and the hydride donor, to afford 4 in excellent yields (92%), after chromatography. Thin-layer chromatography (TLC) analysis, as well as the 1 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). Table 1. Reductive amination of 3: synthesis of the 2,3-diaminopropanol intermediates.  Table 1).

Reagent
The molecular structure of 4 was confirmed by one-and two-dimensional (1D and 2D) NMR spectroscopy. In particular, the 1D 1 H-and 13 C-NMR spectral plots displayed the correct number, multiplicity, and integral values of all resonances assignable to the expected structure of 4. The contour plot of the 2D homonuclear proton TOCSY spectrum of the benzyl derivative 4 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 3.40 ppm, and between 2.60 and 2.90 ppm, which were generated by the resonances of all correlated protons along the molecular backbone of 4 ( Figure 1). Two-dimensional spectroscopy unequivocally 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. The molecular structure of 4 was confirmed by one-and two-dimensional (1D and 2D) NMR spectroscopy. In particular, the 1D 1 H-and 13 C-NMR spectral plots displayed the correct number, multiplicity, and integral values of all resonances assignable to the expected structure of 4. The contour plot of the 2D homonuclear proton TOCSY spectrum of the benzyl derivative 4 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 3.40 ppm, and between 2.60 and 2.90 ppm, which were generated by the resonances of all correlated protons along the molecular backbone of 4 ( Figure 1). Two-dimensional spectroscopy unequivocally 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 2,3diaminopropanols. 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 2,3diaminopropanol scaffold containing the acid-labile benzhydryl masking group, which is orthogonal   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 3 with benzhydrylamine afforded the corresponding protected 2,3-diaminopropanol 5, 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 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 [63,64]. Solvent, temperature, and concentration can affect the free energy 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 5, the minor conformer was 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 2 group is a structural feature displayed by many biologically active compounds [68,69]. Reductive amination of 3 with n-propylamine (Scheme 1, Table 1) gave 8 in an 89% yield after chromatography. The reaction represented a short route to the direct alkylation of the 3-NH 2 function of the 2,3-diaminopropanol scaffold, and the n-propyl derivative 8 was considered to be a possible precursor in the synthesis of alkylated l-Dap structures.

Analysis of Racemization
Chiral α-amino aldehydes show the tendency to racemize under acidic or basic conditions, 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 1-methylphenylamine. Reactions gave the corresponding diastereomeric 2,3-diaminopropanols 6 and 7 (Scheme 2, Table 1), which were obtained in a 90% and 91% yield, respectively, after chromatography. Both pure compounds 6 and 7 exhibited the expected molecular structure, as established by 1 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 1. The absence of racemization 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).
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 1. The absence of racemization 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).

Reductive Amination of the Aldehyde 3 with Sulfonamides
We focused our attention on the reductive amination of 3 with p-toluenesulfonamide and 4-nitrobenzenesulfonamide (Scheme 2, Table 1). The required nucleophiles were prepared from the corresponding commercial sulfonyl chlorides, via reaction with a 20% ammonia solution. Reductive amination of 3 produced the arylsulfonyl-protected 2,3-diaminopropanols 9 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 9 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).
Molecules 2020, 25, x 7 of 20 We focused our attention on the reductive amination of 3 with p-toluenesulfonamide and 4nitrobenzenesulfonamide (Scheme 2, Table 1). The required nucleophiles were prepared from the corresponding commercial sulfonyl chlorides, via reaction with a 20% ammonia solution. Reductive amination of 3 produced the arylsulfonyl-protected 2,3-diaminopropanols 9 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 9 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). 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]. Compound 11 was reputed to be a possible intermediate for the synthesis of N β -methylated L-Dap scaffolds, after transformation of the OH function into a carboxyl group, followed by the final removal of the arylsulfonyl protection.

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. 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]. Compound 11 was reputed to be a possible intermediate for the synthesis of N β -methylated l-Dap scaffolds, after transformation of the OH function into a carboxyl group, followed by the final removal of the arylsulfonyl protection.

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.
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]. Compound 11 was reputed to be a possible intermediate for the synthesis of N β -methylated L-Dap scaffolds, after transformation of the OH function into a carboxyl group, followed by the final removal of the arylsulfonyl protection.

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.

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 3-NH 2 functions, respectively. We were attracted by the idea of masking the side-chain amino function 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.
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. The conversion of 4 into 20 began with the acidolysis of the tert-butyl ether linkage, to generate 15. Unlike the synthesis of methyl ester 14, we preferred to directly oxidize the crude intermediate 2,3-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 Fmoc protecting group. Unblocking of the 2-NH 2 function afforded the carboxylic acid 17, which was subsequently reacted with di-tert-butyl dicarbonate (Boc 2 O) to yield the Boc-derivative 18. A catalytic transfer hydrogenation step [77] was applied to remove the benzyl residue from the 3-NH 2 function of 18, 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 H-and 13 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.

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).
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.

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). This experiment was performed in order to verify the possible use of 21 in solution-phase Fmocchemistry-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-NH2 group, and then separately coupled 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. 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 2 group, and then separately coupled 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.

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 rotations were measured in a 1-dm 3 (2-mL volume) tube, in methanol (c 1.0), and [α] D values were determined at 20 • C, using a digital polarimeter. All NMR spectra were recorded at 25 ± 0.1 • C, using 5-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 1 H-and 13 C-NMR spectra were recorded at 300.132 MHz and 75.03 MHz, respectively. Deuterated chloroform for spectroscopy (CDCl 3 , 99.5% isotopically pure, containing 0.1% tetramethylsilane (TMS)) was used 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 coupling constants (J) are reported in Hz. Relaxation times (T 1 ) for all 1 H and 13 C carbon nuclei 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 respective protons. Two-dimensional homonuclear correlation spectra ( 1 H, 1 H-TOCSY) were obtained by the M.Levitt's composed pulse decoupling sequence (MLEV) for isotropic conditions, with an 80 ms spin-lock period, elaborating spectral data as previously reported [80]. Acquisition and elaboration parameters for one-dimensional 1 H and 13 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 [83,84]. Reactions were monitored by thin-layer chromatography (TLC), using pre-coated silica gel glass plates (Merck, 60-F254, 0.25 mm). The best elution of carboxylic acids was obtained by adding glacial acetic acid (three 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 2 SO 4 ), and H 2 SO 4 (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 2 ). Diazomethane in DCM (0.66 M solution) was freshly prepared 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. The solvent was removed under reduced pressure condition, and the recovered solid residue was re-dissolved in 5% aqueous NaHSO 4 (10 mL), then extracted with diethyl ether (3 × 10 mL). The organic layers were washed with 5% aqueous NaHCO 3 (3 × 10 mL) and brine (3 × 10 mL), dried over Na 2 SO 4 , paper filtered, and evaporated to dryness under reduced pressure conditions. The

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) 4 (0.34 mL, 1.12 mmol) 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 3 CN (52.8 mg, 0.84 mmol) was added. 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.

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 2 CO 3 (CAUTION) until complete gas evolution, and then desalted by elution with small volumes of DCM on a short silica gel pad. The organic solution was dried over Na 2 SO 4

Preparation of Fmoc-l-Dap(Ts)-OCH 3 (14)
Compound 12 (232.8 mg, 0.5 mmol) was dissolved in acetone (5 mL), and aqueous 15% solution of NaHCO 3 (3 mL) was added, followed by solid NaBr (10.2 mg, 0.1 mmol). TEMPO (7.5 mg, 0.05 mmol) was added to the mixture, under magnetic stirring at 0 • C. After slow addition of TCCA (220.5 mg, 5.0 mmol) within 20 min at 0 • C, the mixture was maintained under magnetic stirring overnight at 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 2 CO 3 . The aqueous phase was washed with small portions of AcOEt (3 × 10 mL), treated with 5% aqueous KHSO 4 (10 mL), and re-extracted with AcOEt (3 × 10 mL). The organic layers were dried over MgSO 4 and paper-filtered, and the solvent was evaporated to dryness, yielding a raw material 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,  Step 1: Acidolysis. To a solution of 4 (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 15 min (TLC: diethyl ether/petroleum ether 80:20, v/v). After complete conversion of 4, the mixture was evaporated to dryness under reduced pressure conditions. The residue was dissolved in DCM (5 mL), treated with solid Na 2 CO 3 until complete gas evolution, and desalted on a short pad of Celite 545 ® by elution with small volumes of DCM. The organic phase was dried over Na 2 SO 4 and evaporated to 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 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 2 O (5 mL), containing NaHCO 3 (two equivalents with 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 4 , before being extracted with ethyl acetate (3 × 10 mL). The organic layers were collected, washed once with brine (10 mL), dried over Na 2 SO 4 , paper-filtered, and evaporated to dryness to give a white solid which was transformed in a white powder by co-evaporation from a 2:1 (v/v) methyl-tert-butyl ether (MTB)/n-hexane mixture. TLC (CHCl 3 /MeOH 90:10, v/v): R f = 0.34.
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 2 O/EtOAc mixture (10 mL). The aqueous phase was separated, back extracted with EtOAc (3 × 10 mL), concentrated under reduced pressure conditions, and lyophilized.
Step 6: Protection with Fmoc group. The crude product obtained from step 5 was dissolved in a 1:1 (v/v) 1,4-dioxane/H 2 O mixture (15 mL) containing NaHCO 3 (two equivalents with respect to 4; 88 mg, 1 mmol). A solution of Fmoc-OSu (1.1 equivalents with respect to 4; 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 3 /MeOH 95:5, v/v; ninhydrin as the visualizing agent), and, after complete disappearing of the free amino compound, the solution was concentrated under vacuum, and the aqueous residue was made acidic (pH = 2.5) by adding 5% aqueous KHSO 4 and extracted with ethyl acetate (3 × 10 mL). The collected organic layers were washed once with brine (10 mL), dried over Na 2 SO 4 , paper=filtered, 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.  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 × 5 mL), then dried under vacuum. The recovered crude product 22 was dissolved in DCM (3 mL) and added to a magnetically stirred mixture of DIEA (0.35 mL; 2.03 mmol), HOBt monohydrate (150 mg; 0.97 mmol), and EDC (160 mg; 0.85 mmol) in DCM (5 mL). 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 3 (5 mL), once with 5% aqueous KHSO 4 (5 mL), and once with brine (5 mL). The organic layer was dried over Na 2 SO 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).

Conclusions
In summary, a synthetic route is proposed for the multi-step preparation of orthogonal di-protected l-Dap methyl esters. In the proposed procedure, commercial N α -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.