New synthetic strategy for o-NBS protected amino acids and their use in synthesis of mono-benzylated peptides

Article abstract of DOI:10.1016/j.tetlet.2005.07.154

A synthetic strategy to prepare o-NBS protected Fmoc-amino acids under mild conditions, in a rapid and efficient way, characterised by high yields and excellent purity of the final products has been developed. The o-NBS protected Fmoc-amino acids are employed in solid phase peptide synthesis to prepare peptidomimetics carrying mono-benzylated moieties on peptide side chains.

Full text of DOI:10.1016/j.tetlet.2005.07.154

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 6637–6640
New synthetic strategy for o-NBS protected amino acids
and their use in synthesis of mono-benzylated peptides
Stefania De Luca, Raffaella Della Moglie, Antonia De Capua and Giancarlo Morelli*
Department of Biological Sciences, Biostructures Section, & CIRPeB, University of Naples ‘Federico II’,
via Mezzocannone, 16 Naples I-80134, Italy
Received 14 June 2005; revised 27 July 2005; accepted 29 July 2005
Available online 15 August 2005
Abstract—A synthetic strategy to prepare o-NBS protected Fmoc-amino acids under mild conditions, in a rapid and efficient way,
characterised by high yields and excellent purity of the final products has been developed. The o-NBS protected Fmoc-amino acids
are employed in solid phase peptide synthesis to prepare peptidomimetics carrying mono-benzylated moieties on peptide side chains.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The choice of amine protecting groups is critical for the
chemical synthesis of amino acid derivatives and of
bioorganic compounds in general. Among the different
possibilities,1 the 2-nitrobenzenesulfonamide (o-NBS)
group as received increasing attention.2–6 As a protect-
ing group for primary and secondary amines, it is stable
under both acidic and basic conditions. Moreover, its
attachment to primary amines greatly increases the acid-
ity of the amine hydrogen enough to allow selective
amine mono-alkylations via Mitsunobu conditions in
addition to more traditional approaches (K2CO3,
halide).2
propionic acid (Dap), in which the benzyl moiety is at
different distances from the peptide backbone. In fact,
rational-designed peptidomimetics often contain no nat-
ural amino acids able to fit the hydrophobic binding
pocket of macromolecular receptors. By changing the
length of amino acidic side chains, it is possible to
accommodate the ligand in the binding site of the recep-
tor and meet the topochemical array of the pharmaco-
phore model.
The aim of this work has been to develop a synthetic
strategy to synthesise Fmoc-protected amino acids
(Fmoc = 9-fluorenylmethoxycarbonyl) suitable to pre-
pare mono-benzylated peptides. Fmoc-amino acid
derivatives containing 2-nitrobenzenesulfonamide on
the side-chain amino function result in very attractive
building blocks to use in solid phase peptide and pepti-
domimetic synthesis.
As the 2-nitrobenzenesulfonamide group is stable under
standard amino acid/peptide chemistry protocols, both
in solution and on solid phase,7 it can also be a suitable
protecting group for amino functions of amino acid
derivatives used in peptide or peptidomimetic synthesis.
When bound to the side-chain amino group, 2-nitrobenz-
enesulfonamide group remains stable during peptide
assembly and can be removed at the end of the peptide
synthesis. Moreover, it allows the mono-alkylation dur-
ing the peptide chain elongation in solid phase. For
example, by using this strategy, it can be possible to ob-
tain peptidomimetics, which contain amino benzyl
derivatives of several amino acid residues, such as lysine,
ornithine (Orn), di-aminobutyric acid (Dab) or di-amino-
Although synthetic methods for amine protection by
using 2-nitrobenzensulfonyl chloride are well estab-
lished,5 the introduction of o-NBS onto amino acid
amine function in aqueous solution of NaOH 1 N8,9 is
incompatible with the presence of a basic labile protect-
ing group such as Fmoc.
Herein, we describe a novel and efficient procedure to
obtain Fmoc-amino acid derivatives carrying o-NBS
group on the side-chain amino group. Moreover, the
use of these residues in solid phase peptide synthesis to
prepare amino mono-benzylated peptides is also
reported.
Keywords: o-NBS protected amino acids; Mono-benzylated peptides;
Building blocks for peptide synthesis.
*
Corresponding author. Tel.: +39 081 253 6650; fax: +39 081 253
4574; e-mail: morelli@chemistry.unina.it
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.07.154

6638
S. De Luca et al. / Tetrahedron Letters 46 (2005) 6637–6640
Standard procedures for the introduction of o-NBS
group onto primary or secondary amines in good yield
consist of the reaction of o-NBS chloride in organic sol-
vent, such as dichloromethane and dimethylformamide,
in the presence of an organic base (triethylamine, di-iso-
propylethylamine or pyridine).5 These methods are
hardly suitable in the case of Fmoc-amino acid deriva-
tives containing an amine function on the side chain
and a free carboxylic function such as Fmoc-Lys-OH,
Fmoc-Orn-OH, Fmoc-Dab-OH and Fmoc-Dap-OH. It
was previously reported that the introduction of o-
NBS as an amino protecting group onto amino acids
in organic solvent requires the amino acids to be used
as methyl ester derivatives.7 In fact, the amphipatic
property of the o-NBS protected derivatives, due to
the presence of aromatic groups (Fmoc and o-NBS)
and of free carboxylic function, makes the separation
of the desired product from the organic ammonium salt
and the final chromatographic purification difficult, thus
giving a very low yield of the pure product.
Fmoc-NH-CH-COOH
CH2
Fmoc-NH-CH-COOH
CH2
(b)
(a)
NH
CH2
NH
o-NBS
o-NBS
2) 1N HCl
K2CO3 in H2O
2) 1N HCl
1) 10% K2CO3
in H2O/ Dioxane
(4/1)
1) 10%
NO2
Fmoc-NH-CH-COOH
+
(CH2)
n
SO2Cl
NH2
1eq
r.t., 4 h
1) 10% K2CO3
in H2O/Dioxane
(1/1)
1) 10% K2CO3
in H2O/Dioxane
(2/5)
We propose a synthetic strategy to prepare Fmoc-Lys-
(o-NBS)-OH, Fmoc-Orn(o-NBS)-OH, Fmoc-Dab(o-NBS)-
OH and Fmoc-Dap(o-NBS)-OH, which avoids the
synthetic problems described above in the standard
procedures.
2) 1N HCl
2) 1N HCl
Fmoc-NH-CH-COOH
Fmoc-NH-CH-COOH
CH2
CH2
CH2
CH2
CH2
Our method of synthesis uses potassium carbonate
aqueous solution or a mixture of this solution and 1,4-
dioxane as a system solvent to protect the side-chain
amine with o-NBS group. Mild conditions and inexpen-
sive reagents are used to obtain the desired compounds
in excellent yield and purity.
(c)
(d)
CH2
NH
CH2
NH
o-NBS
o-NBS
In Scheme 1, the synthetic routes employed to obtain
each o-NBS protected Fmoc-amino acid derivatives
are summarised in detail. After dissolving the Fmoc-
amino acid derivative in 10% aqueous solution of
K2CO3 and, for some of them, a small amount of 1,4-
dioxane, the mixture is stirred in an ice-water bath and
1 equiv of o-NBS chloride is added in small portion.
The resulting solution is stirred at room temperature
for about 4 h. Any unreacted o-NBS chloride is easily re-
moved by extraction with diethyl ether and the final
product is recovered as a pale-yellow precipitate by acidi-
fying the aqueous layer with 1 N HCl. Any unreacted
Fmoc-amino acid remains soluble in the acidic aqueous
solution, while the carbonate is completely neutralised.
By using this strategy, the o-NBS protected Fmoc-ami-
no acid derivatives are collected in high yield and in
excellent purity,10 as assessed by analytical RP-HPLC,
(a) Fmoc-Dap(o-NBS)-OH, yield 84 %, purity 98 %;
(b) Fmoc-Dab(o-NBS)-OH, yield 65 %, purity 98 %;
(c) Fmoc-Orn(o-NBS)-OH, yield 64 %, purity 98 %;
(d) Fmoc-Lys(o-NBS)-OH, yield 80 %, purity 98 %;
Scheme 1. Synthetic routes to obtain o-NBS protected Fmoc-amino
acid derivatives.
procedure to introduce o-NBS onto side-chain amine
groups of a peptide chain bound to a solid support is
supposed to employ expensive Fmoc-amino acid deriva-
tives. In fact, such derivatives contain an orthogonal
amine protecting group, which is selectively removed
during the peptide assembly, in order to allow the intro-
duction of o-NBS group.
1
LC-ES-MS and H NMR spectroscopy, and any chro-
matographic purification is not needed.11 In Scheme 1,
yield and purity data for the four amino acid derivatives
prepared are reported, while their full characterisation is
summarised in the references and notes.12
It is clear that the use of o-NBS protected Fmoc-amino
acid derivatives as building blocks in peptide synthesis
simplifies the procedure to synthesise mono-alkylated
peptide derivatives under conventional conditions
(K2CO3, halide). Such a modification of peptides is par-
ticularly interesting, since it could allow the introduction
of conformational constraints desired in rational design
approach. On this regard, solid phase preparation of
mono-benzylated peptides results in a valuable applica-
bility of the N-alkylation proposed procedure.
The proposed synthetic pathway presents additional
advantages when the amino acid derivatives obtained
are intended to be used as building blocks for Fmoc
solid phase peptide synthesis. The introduction of o-NBS
onto amine groups during the solid phase peptide syn-
thesis is a well-established reaction.7 Nevertheless, the

S. De Luca et al. / Tetrahedron Letters 46 (2005) 6637–6640
6639
We synthesised two peptides in which the newly devel-
oped o-NBS protected Fmoc-amino acids were incorpo-
rated into the peptide chain by using Fmoc solid phase
standard methods, as shown in Scheme 2.13 The first
example is given by the synthesis of the nonapeptide
(1) containing in position 8 a lysine residue functional-
ised on the e-NH2 with the benzyl group. Having the
Fmoc-Lys(o-NBS)-OH bound to the growing peptide
chain, the benzylation reaction was performed under
conventional conditions as the next step. After the com-
plete assembly of the peptide onto the solid support the
o-NBS protecting group was removed by using 2-mer-
capto-ethanol in combination with DBU. In order to
verify the absence of any side reactions on the o-NBS
protected amines during peptide elongation and the effi-
ciency of the benzylation reaction performed at the end
of the peptide synthesis, we repeated the synthesis of
(1) by inverting the synthetic steps i and ii. The final com-
pound was isolated and analysed by RP-HPLC and LC-
ES-MS.14 Both strategies provided peptide (1) in similar
yields. The dipeptide H-Lys(Bn)-Gly-OH (2) was iso-
lated during the solid phase peptide synthesis and com-
Fmoc-Gly
Fmoc-Lys(o-NBS)-OH
Fmoc-Lys(o-NBS)-Gly
Fmoc-Dap(o-NBS)-OH
Fmoc-Dap(o-NBS)-Gly
1
pletely characterised by RP-HPLC, LC-ES-MS and H
NMR.15 The mono-benzylated dipeptide was obtained
in almost quantitative yield, which proved the high effi-
ciency of the synthetic procedure employed.
i
i
The second example is another nonapeptide (3) contain-
ing a benzyl group on the b-NH2 of di-aminopropionic
acid introduced in position 8 of the sequence. In this
case, we did not remove the o-NBS group onto the pep-
tide, in order to check its stability under mildly basic
coupling conditions (0.12 M di-isopropylethylamine),
Fmoc removal conditions (20% piperidine), and also
the acidic treatment necessary for cleavage and depro-
tection of the peptide (95% TFA). By ESI mass analysis,
it was assessed that the final peptide was obtained in
high yield and retained the complete amount of the
o-NBS moiety onto the b-NH2 of Dap residue.16
Fmoc-NH-CH-CO-Gly
Fmoc-NH-CH-CO-Gly
(
CH2)4
N
CH2
N
o-NBS
Bn
o-NBS
Bn
iii
ii
iv
In conclusion, we describe a synthetic strategy to pre-
pare o-NBS protected Fmoc-amino acids under mild
conditions, in a rapid and efficient way, characterised
by high yields and excellent purity of the final products.
The o-NBS protected Fmoc-amino acids are very useful
building blocks to be employed in solid phase peptide
synthesis, since the o-NBS group is compatible with
many protecting groups widely used in peptide/organic
synthesis. Their use allows the synthesis of peptidomi-
metics modified with mono-benzylated functions on
peptide side chains, without any modification of the syn-
thesis protocols.
iv
H-Lys(Bn)-Gly-OH
2
H-Asp-Tyr-Met-Dap-Trp-Met-Asp-Dap(Bn, o-NBS)-Gly-OH
3
ii
iii
iv
Acknowledgements
This work was supported by funds from Italian Minister
of Research (MIUR) and Italian Council of Research
(CNR), Progetto Strategico: Oncologia. We thank Luca
De Luca for technical support.
H-Asp-Tyr-Met-Dap-Trp-Met-Asp-Lys(Bn)-Gly-OH
1
References and notes
Scheme 2. Peptide synthesis according to the Fmoc solid phase
protocol. Reagents and conditions: Wang resin, loading of
0.99 mmol gꢀ1; (i) K2CO3 (2 equiv), benzyl bromide (2 equiv), in
DMF, 1 h; (ii) protected amino acids, PyBop/HOBt/DIPEA, in DMF,
1 h; (iii) DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (10 equiv), 2-mer-
capto-ethanol (5 equiv), in DMF, 30 min; (iv) TFA/H2O/TIS (TIS =
tris-iso-propylsilane) (97:2:1), 1.5 h. Synthesis of (1) can also be
obtained by inverting steps i and ii.
1. (a) Sandler, R. Organic Functional Group Preparations,
2nd ed.; Academic Press: New York, 1983, Chapter 13; (b)
Marson, C. M.; Hobson, A. D. In Comprehensive Organic
Functional Group Transformation; Katritzky, A., Meth-
Cohn, O., Rees, C. W., Eds.; Pergamon Press: Oxford,
1995; Vol. 2, p 297.

6640
S. De Luca et al. / Tetrahedron Letters 46 (2005) 6637–6640
2. Fukuyama, T.; Jow, C. K.; Cheung, M. Tetrahedron Lett.
H1,4,5,8), 7.53 (d, J = 7.3 Hz, 1H, NH), 7.42 and 7.33 (2t,
J = 7.3 Hz, 4H, Fmoc-H2,3,6,7), 4.26 (d, J = 6.5 Hz, 2H,
Fmoc-CH2), 4.22 (t, J = 6.7 Hz, 1H, Fmoc-CH), 3.98 (m,
1H, Dap-CHa), 3.3 (overlaps HDO signal, 2H, Dap-
CH2b); ES-MS: calcd (M+H+), 512.1; found, m/z 512.3;
HS-MS: calcd (M+H+ = C24H22O8N3S+), 512.1128;
1995, 36, 6373–6374.
3. Fukuyama, T.; Cheung, M.; Jow, C. K.; Hidai, Y.; Kan,
T. Tetrahedron Lett. 1997, 38, 5831–5834.
4. Hidai, Y.; Kan, T.; Fukuyama, T. Tetrahedron Lett. 1999,
40, 4711–4714.
5. Kurosawa, W.; Kan, T.; Fukuyama, T. Org. Synth. 2002,
79, 186–195.
20
found, m/z 512.1135; ½aꢂD ꢀ15.5 (c 1.05, CH3OH).
Na-(9-Fluorenylmethoxycarbonyl)-Nc-2-nitrobenzen-sulfon-
yl-L-2,4-di-aminobutyric acid (b). HPLC: tR = 15.57 min
6. Kan, T.; Fukuyama, T. Chem. Commun. 2004, 235–
354.
1
(gradient 2); H NMR (500 MHz, DMSO-d6): d 8.23 (br,
7. Reichwein, J. F.; Liskamp, R. M. J. Tetrahedron Lett.
1998, 39, 1243–1246.
8. Bodanszky, M.; Bodanszky, A. The Practice of Peptide
Synthesis, 2nd ed.; Springer: New York, USA, 1994,
Chapter 2.
1H, o-NBS-NH), 7.96 and 7.83 (2m, 4H, o-NBS-H3,4,5,6),
7.88 and 7.69 (2d, J = 7.3 Hz, 4H, Fmoc-H1,4,5,8), 7.60 (d,
J = 7.2 Hz, 1H, NH), 7.40 and 7.30 (2t, J = 7.3 Hz, 4H,
Fmoc-H2,3,6,7), 4.25 (d, J = 6.5 Hz, 2H, Fmoc-CH2), 4.21
(t, J = 6.7 Hz, 1H, Fmoc-CH), 3.98 (br, 1H, Dab-CHa),
c
9. Di Gioia, M. L.; Leggio, A.; Liguori, A. J. Org. Chem.
2005, 70, 3892–3897.
2.97 (m, 2H, Dab-CH2 ), 1.91 and 1.77 (2m, 2H, Dab-
CH2b); ES-MS: calcd (M+H+), 526.1; found, m/z 526.3;
10. General procedure for the synthesis of (a), (b), (c) and (d).
Approximately 1 mmol of each Fmoc-amino acid was
suspended in a mixture of 10% aqueous solution of K2CO3
and 1,4-dioxane. To dissolve Fmoc-Dab-OH, Fmoc-Orn-
OH and Fmoc-Lys-OH the following volume (mL) of 10%
bicarbonate in water/1,4-dioxane were used, respectively:
4:1, 4:4 and 2:5; 4.5 mL of 10% bicarbonate in water was
used only to dissolve Fmoc-Dap-OH. The solution was
stirred and cooled in an ice-water bath while 0.9 equiv
(ꢁ200 mg) of o-nitrobenzensulfonyl chloride, dissolved in
a small portion of dioxane (150 lL), was added over a
period of 5 min. The stirring was continued at room
temperature for about 4 h. The resulting reaction mixture
was than poured into water (80 mL) and extracted with
diethyl ether (40 mL in two portions). The organic layer
was washed twice with basic water to recover some of the
final product, which had passed into the ether. TLC
analysis (eluent: dichloromethane/methanol, 9:1) of the
aqueous layer showed a quite clean formation of the final
protected product (d) (Rf = 0.2), with only a small spot of
the Fmoc-amino acid on the TLC start line. The aqueous
solution is cooled in an ice-water bath and acidified under
vigorous stirring with 1 N HCl in water. This allows to
precipitate the desired compound (d) as a pale-yellow
solid. After 1 h, the mixture is filtered and the dry product
was isolated with high purity, as assessed by HPLC and
1H NMR analysis.
11. Analytical RP-HPLC runs were carried out on a HP
Agilent Series 1100 apparatus using a Phenomenex (Tor-
rance, CA) C18 column, 4.6 · 250 mm with a flow rate of
1.0 mL minꢀ1. Preparative RP-HPLC was carried out on a
Shimadzu 8A apparatus equipped with an UV Shimadzu
detector using a Phenomenex (Torrance, CA) C18 column,
22 · 250 mm with a flow rate of 20 mL minꢀ1. The system
solvent used was H2O 0.1% TFA (A) and CH3CN 0.1%
TFA (B), with a linear gradient from 5% to 70% B in
30 min (gradient 1) or from 30% to 95% B in 20 min
(gradient 2). LC-ES-MS data were obtained using a
Finnigan Surveyor MSQ single quadrupole electrospray
ionisation mass spectrometer coupled with a Finnigan
Surveyor HPLC (Finnigan/Thermo Electron Corporation
San Jose, CA, USA). HR-ES-MS were run on a Micro-
mass QTOF mass spectrometer. 1H NMR spectra were
recorded on a Varian INOVA-Unity 500 MHz spectro-
meter. Specific rotation (in deg mL/g dm) is measured by
using a Perkin–Elmer model 141 polarimeter.
HS-MS: calcd (M+H+ = C25H24O8N3S+), 526.1284;
20
found, m/z 526.1291; ½aꢂD ꢀ7.2 (c 1.04, CH3OH).
Na-(9-Fluorenylmethoxycarbonyl)-Nd-2-nitrobenzen-sulfon-
1
yl-L-ornithine (c). HPLC: tR = 15.83 min (gradient 2); H
NMR (500 MHz, DMSO-d6): d 8.12 (br, 1H, o-NBS-NH),
7.96 and 7.83 (2m, 4H, o-NBS-H3,4,5,6), 7.89 and 7.70 (2d,
J = 7.3 Hz, 4H, Fmoc-H1,4,5,8), 7.51 (d, J = 7.5 Hz, 1H,
NH), 7.40 and 7.31 (2t, J = 7.3 Hz, 4H, Fmoc-H2,3,6,7),
4.26 (d, J = 6.8 Hz, 2H, Fmoc-CH2), 4.20 (t, J = 6.8 Hz,
1H, Fmoc-CH), 3.84 (m, 1H, Orn-CHa), 2.88 (t, 2H,
c
Orn-CH2 ), 1.71 and 1.56 (2m, 2H, Orn-CH2b),1.48 (m,
d
2H, Orn-CH2 ); ES-MS: calcd (M+H+), 540.1; found, m/z
540.2;
HS-MS:
calcd
(M+H+ = C26H26O8N3S+),
20
540.1441; found, m/z 540.1449; ½aꢂD ꢀ3.4 (c 1.03, CH3OH).
Na-(9-Fluorenylmethoxycarbonyl)-Ne-2-nitrobenzen-sulfon-
yl-L-lysine (d). HPLC: tR = 16.26 min (gradient 2); 1H
NMR (500 MHz, DMSO-d6): d 8.12 (br, 1H, o-NBS-NH),
7.97 and 7.86 (2m, 4H, o-NBS-H3,4,5,6), 7.89 and 7.72 (2d,
J = 7.3 Hz, 4H, Fmoc-H1,4,5,8), 7.60 (d, J = 7.8 Hz, 1H,
NH), 7.41 and 7.32 (2t, J = 7.3 Hz, 4H, Fmoc-H2,3,6,7),
4.28 (d, J = 6.8 Hz, 2H, Fmoc-CH2), 4.22 (t, J = 6.8 Hz,
1H, Fmoc-CH), 3.87 (m, 1H, Lys-CHa), 2.88 (m, 2H,
e
Lys-CH2 ), 1.64 and 1.56 (2m, 2H, Lys-CH2b), 1.42
d
c
(m, 2H, Lys-CH2 ), 1.31 (m, 2H, Lys-CH2 ); ES-MS: calcd
(M+H+), 554.1; found, m/z 554.0; HS-MS: calcd
(M+H+ = C27H28O8N3S+),
554.1597;
found,
m/z
20
554.1589; ½aꢂD ꢀ2.7 (c 1.02, CH3OH).
13. The peptides synthesis was carried out by solid phase
method using the standard Fmoc procedure. The first
amino acid was loaded on Wang resin according to
literature protocol (Sheppard, R. C.; Williams, B. J. Int. J.
Peptide Protein Res. 1982, 20, 451–454) and the loading
was evaluated by Fmoc test. Amino acid couplings were
monitored by Kaiser test (Kaiser, E.; Colescott, R. L.;
Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970, 34,
595–598).
14. H-Asp-Tyr-Met-Dap-Trp-Met-Asp-Lys(Bn)-Gly-OH (1).
Yield: 32–36%; HPLC: tR = 27.35 min (gradient 1); ES-
MS: calcd (M+H+=C56H76O15N12S2+), 1220.5; found,
m/z 1220.5.
15. H-Lys(Bn)-Gly-OH (2): Yield: 78%; HPLC: tR
=
13.52 min (gradient 1); 1H NMR (500 MHz, D2O): d
7.50–7.41 (m, 5H, Lys-ArH), 4.22 (m, 2H, Gly-CH2a),
3.95–3.88 (2m, 3H, Lys-CH2-Ar and Lys-CHa), 2.95 (t,
e
J = 7.6 Hz, 2H, Lys-CH2 ), 1.91 (m, 2H, Lys-CH2b), 1.66
d
c
(m, 2H, Lys-CH2 ), 1.44 (m, 2H, Lys-CH2 ); ES-MS: calcd
(M+H+=C15H23O3N3+), 294.2; found, m/z 294.6.
16. H-Asp-Tyr-Met-Dap-Trp-Met-Asp-Dap(Bn, o-NBS)-Gly-
OH (3). Yield: 35%; HPLC: tR = 31.31 min (gradient 1);
ES-MS: calcd (M+H+=C59H74O19N13S3+), 1364.4; found,
m/z 1364.6.
12. Na-(9-Fluorenylmethoxycarbonyl)-Nb-2-nitrobenzen-sulfon-
yl-L-2,3-di-aminopropionic acid (a). HPLC: tR
=
1
15.49 min (gradient 2); H NMR (500 MHz, DMSO-d6):
d 8.20 (br, 1H, o-NBS-NH), 7.98 and 7.85 (2m, 4H, o-
NBS-H3,4,5,6), 7.89 and 7.71 (2d, J = 7.3 Hz, 4H, Fmoc-