4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium Toluene-4-sulfonate (DMT/NMM/TsO?) Universal Coupling Reagent for Synthesis in Solution

Article abstract of DOI:10.1002/hlca.201700187

4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TsO^?), a representative member of the inexpensive and environmentally-friendly N-triazinylammonium family of sulfonates, has been found to be a very effective coupling reagent for the synthesis of amides, esters and peptides in solution. This study confirms the usefulness of DMT/NMM/TsO^? for peptide synthesis in solution, starting from Z-, Fmoc-, and Boc-protected substrates as well as unnatural building blocks. Peptide synthesis with DMT/NMM/TsO^? produced high yields, with high crude product purity and low risk of racemization. In all cases, stoichiometric amounts of reagents were used and the standard synthetic procedure, without the need for time-consuming optimization stages or expensive chromatographic purification. DMT/NMM/TsO^? was also found to be very useful for the synthesis of oligopeptides using a fragment coupling strategy.

Full text of DOI:10.1002/hlca.201700187

Article Type: Full Paper
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
Toluene-4-sulfonate (DMT/NMM/TsO-) Universal Coupling Reagent
for Synthesis in Solution
Justyna Fraczyk,a Zbigniew J. Kaminski,a Joanna Katarzynska,a and Beata Kolesinska*,a
a Institute of Organic Chemistry, Lodz University of Technology, 90-924 Lodz, Poland,
beata.kolesinska@p.lodz.pl
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TsO-),
a representative member of the inexpensive and environmentally-friendly N-triazinylammonium
family of sulfonates, has been found to be a very effective coupling reagent for the synthesis of
amides, esters and peptides in solution. This study confirms the usefulness of DMT/NMM/TsO- for
peptide synthesis in solution, starting from Z-, Fmoc- and Boc-protected substrates as well as
unnatural building blocks. Peptide synthesis with DMT/NMM/TsO- produced high yields, with high
crude product purity and low risk of racemization. In all cases, stoichiometric amounts of reagents
were used and the standard synthetic procedure, without the need for time-consuming optimization
stages or expensive chromatographic purification. DMT/NMM/TsO- was also found to be very useful
for the synthesis of oligopeptides using a fragment coupling strategy.
Keywords: triazine-based coupling reagent • peptide • ester • coupling of fragments • standard
protocol
Introduction
A wide range of coupling reagents is available for peptide synthesis [1-9]. Nevertheless, synthetic
methods require further improvement to facilitate difficult coupling, involving less reactive or
sterically-hindered substrates. It is also necessary to reduce the costs of peptide manufacturing,
isolation and purification and to eliminate hazardous or environmentally harmful components,
procedures and side products.
In this study, an innovative strategy was developed for improving coupling. Excessive over-activation
of the acylating component, which usually elicits a side reaction, was avoided. At the same time,
high effectiveness was achieved by introducing into the synthetic pathway leading to the formation
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/hlca.201700187
This article is protected by copyright. All rights reserved.

of amide or ester bonds an additional, synchronous and thermodynamically highly favored process,
involving rearrangement of the side product.
The goal was reached by designing the “superactive ester” 4 [10] giving activation “push” by electron
withdrawing effect of 1,3,5-triazine ring [11], amplified by the synchronous “pull” caused by
additional stabilization of the strongly acidic 2-hydroxy-1,3,5-triazine 9 side-product through the
fast, highly thermodynamically favored process of intramolecular neutralization by basic nitrogen
atoms of the triazine ring (Scheme 1, pathway II: acylation). Moreover, proceeding via the cyclic 6-
membered transition state, this process is facilitated by the Thorpe-Ingold effect, enhancing
coupling of sterically-hindered substrates.
Scheme 1. Coupling using triazine reagents. In situ synthesis of DMT/NMM/Cl- (3), activation of carboxylic acid to 2-acyloxy-4,6-dimethoxy-1,3,5-triazine (4),
degradation of DMT/NMM/Cl- (3) to inactive coupling reagent compound 4, synthesis of stable DMT/NMM/TsO- (7), intramolecular transfer of the proton
from the tetrahedral intermediate to the triazine nitrogen leading to the thermodynamically-preferred form of the amide function of triazinone 8 leaving
group.
This article is protected by copyright. All rights reserved.

The substrates necessary for the preparation of the “superactive esters” were neither expensive nor
environmentally harmful. The scope of the procedure leading to triazine superactive ester formation
was very broad [12]. However, the multi-stage in situ procedure of triazine superactive ester
formation by treating the carboxylic component with 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT)
in the presence of N-methylmorpholine was complicated to perform. Attempts to simplify the
synthetic procedure by using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride
(3) (DMT/NMM/Cl), identified as a reactive intermediate, were ambiguous (Scheme 1, pathway I).
Kunishima et al. [15] describe 3 (using the acronym DMTMM) as a universal reagent for the synthesis
of peptides and esters [16-20]. However, the efficacy of coupling under standard conditions for
peptide synthesis (in anhydrous DMF) has been found to be unsatisfactory [13], especially in the
case of SPPS [14]. Studies have further revealed that, under anhydrous conditions, quaternary
ammonium chloride DMT/NMM/Cl is prone to demethylation in a process [21] analogous to the Von
Braun reaction [22], yielding methyl chloride and non-reactive 2-(morpholi-4-yl)-4,6-dimethoxy-
1,3,5-triazine (5) (Scheme 1, pathway I). To make the proposed strategy of general use, it was
necessary to eliminate difficulties caused by the insufficient stability of N-triazinylammonium
chloride 3. Substitution of the nucleophilic chloride anion with the less nucleophilic sulphonate
produced a collection of stable coupling reagents with diverse reactivities (Scheme 1, pathway I).
Screening their synthetic potential revealed p-toluenesulphonate 7 to be the most expedient for
SPPS [23]. Because acylating components are used in large excess in SPPS, attempts were made to
investigate the use of 7 under standard conditions in representative syntheses in solution. The utility
of DMT/NMM/TsO- was tested for the synthesis of amides, esters and peptides, including
oligopeptides, using the step by step and fragment coupling strategy. No excess of reagents was
used, and all reactions were performed following standard synthetic procedures, without
optimization stages.
Results and Discussion
An effective coupling reagent should allow the synthesis of various carboxylic acid (amino acid)
derivatives, including amides, esters, thioesters and peptides, with high yields and without losing
optical homogeneity. In the first stage of our study, attempts were made to verify the usefulness of
DMT/NMM/TsO- for amide synthesis. As in the case of other classic reagents, condensation
mediated by 7 occurs in two stages. The first stage involves activation of the carboxyl function of
acid, leading to the formation of the appropriate superactive esters 4. Acylation of the nucleophile
occurs in the second stage. Formation of super-active esters following the treatment of carboxylic
acids with DMT/NMM/TsO- was confirmed by TLC analysis using 4-(4-nitrobenzyl)pyridine (NBP) [24],
which enables visualization of the activation product 4. DMT/NMM/TsO- was been found to be an
effective reagent for the synthesis of amides 10 derived from both aromatic and aliphatic acids and
amines (see Table 1). In all cases, stoichiometric quantities of reagents were used. The reaction
yields were in the range of 88-99% and the purity of the crude amides 10a-e, isolated after a simple
washing procedure, were determined using RP-HPLC as being in the range of 89-99%.
This article is protected by copyright. All rights reserved.

Table 1. Synthesis of Amides 10a-e with DMT/NMM/TsO-.
Entry
Product
Purity [%]
Yield [%]
1.1
O
93
99
H
N
O
10a
1.2
1.3
1.4
1.5
98
95
89
99
92
90
93
88
H
N
O
O
10b
O
N
H
10c
H
N
O
10d
O
N
H
O
NH
10e
O
From a technical standpoint, the very high purity of the final crude products, which were purified
only by washing the reaction mixture with aqueous solutions of NaHCO3 and NaHSO4, is very
important. Our results show that using DMT/NMM/TsO- as a coupling reagent enables costly and
time-consuming chromatographic purification processes to be avoided.
In the next stage of our investigation, we evaluated the utility of DMT/NNM/TsO- for ester 11
synthesis. Acylation of alcohols, which are less nucleophilic than amines, usually requires more
vigorous reaction conditions and the use of more powerful acylating reagents.
Because of their orthogonality, we studied the synthesis of allyl esters of N-protected amino acids
(see Table 2, entry 2.1-2.7). The allyl esters of Fmoc-Asp(OtBu)-OAll and Fmoc-Glu (OtBu)-OAll were
found to be particularly useful for the synthesis of cyclic peptides, producing yields of 86% and 96%,
respectively. The results of comparative studies on the synthesis of allyl esters of Fmoc-Asp (OtBu)-
OAll with classical carbodiimide, uronium and phosphonium salts coupling reagents (Table 2, entry
2.8.-2.11) clearly indicated the usefulness and the advantage of DMT/NMM/TsO- as a coupling
reagent in the synthesis of esters in solution. Synthesis of active esters (Pfp - pentafluorophenyl and
NSu - N-hydrosycuccinimide) also occurred in the presence of DMT/NMM/TsO-, with satisfactory
yields in the range of 77-88%.
This article is protected by copyright. All rights reserved.

Table 2. Synthesis of esters 11a-n of carboxylic acids using DMT/NNM/TsO- as a coupling reagent.
Entry
Ester
Yield [%]
Purity [%]
%L / %D a
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
Allyl ester of 4-methoxybenzoic acid (11a)
Fmoc–L-Ala-OAll (11b)
82
86
87
77
82
96
86
70
99
88
95
93
93
96
99
92
-
100/0
99.5/0.5
100/0
99.6/0.4
99.5/0.5
100/0
-
Fmoc–L-Phe-OAll (11c)
Fmoc-L-Glu-(OtBu)-OAll (11d)
Fmoc-L-Ser(tBu)-OAll (11e)
Fmoc-L-Lys(Boc)-OAll (11f)
Fmoc-L-Asp-(OtBu)-OAll (11g)
Fmoc-L-Asp-(OtBu)-OAll (11g)
by using DCC
2.9
Fmoc-L-Asp-(OtBu)-OAll (11g)
70
72
59
99
98
99
-
-
-
by using TBTU
2.10
2.11
Fmoc-L-Asp-(OtBu)-OAll (11g)
by using HATU
Fmoc-L-Asp-(OtBu)-OAll (11g)
by using BOP
2.12
2.13
2.14
2.15
2.16
2.17
2.18
Fmoc-L-Asp-(OtBu)-OPfp (11h)
Fmoc-L-Ala-OPfp (11i)
88
83
78
94
90
88
80
93
88
95
97
96
95
97
100/0
100/0
100/0
100/0
99/1
Fmoc-L-Ala-ONSu (11j)
Fmoc-L-Ala-SPh (11k)
Fmoc-L-Ser(tBu)-SPh (11l)
Fmoc-L-Ala-S(CH2)3CH3 (11m)
Fmoc-L-Ser(tBu)-S(CH2)3CH3 (11n)
99/1
98/2
a Enantiomeric purities were determined by GC analysis on a ChirasilVal column after degradation of peptides to amino acids and subsequent formation of
volatile N-Tfa-amino acid-OMe.
The versatility of DMT/NMM/TsO- for the synthesis of thioesters of N-protected amino acids was
especially interesting. In all cases, the yields and purity of the crude products were exceptionally
high. It should be emphasized that, regardless of both the structure of the activated amino acid and
of the thiol used, no racemization of the final product was observed. During the derivatization
protocol, racemization may occur under acidic hydrolysis, resulting in low D enantiomer content.
In the next stage of our research, we investigated the utility of DMT/NMM/TsO- for peptide
synthesis in solution. It had been demonstrated that DMT/NMM/TsO- can be very efficient for the
synthesis of orthogonally-protected dipeptides 12 with Z, Boc and Fmoc protecting groups, derived
from a broad range of natural and unnatural hindered amino acids (Table 3). In solution, all
protected dipeptides were obtained with very high purity, evaluated by LC-MS analysis.
Homogeneous products were obtained, isolated and purified by simple extraction, including in the
cases of the sterically-hindered aminoisobutyric acid derivatives, and of serine and threonine with an
O-protected in the side chain by a bulky t-Bu group. The yields of the reaction ranged from 70-98%,
and purity from 88-99% (see Table 3).
This article is protected by copyright. All rights reserved.

Table 3. Dipeptides 12a-u synthesized in solution with DMT/NMM/TsO-.
Entry
Dipeptide
Yield [%]
Purity [%]
%L / %D a
N-term.
C-term
3.1
Z-Aib-L-Ala-OMe (12a)
85
81
84
74
71
77
74
70
73
87
87
91
88
98
77
95
99
99
80
90
89
93
94
86
84
96
98
98
99
98
-
100/0
100/0
100/0
99/1
3.2
Z-Aib-L-Phe-OMe (12b)
-
3.3
Z-Aib-L-Leu-OMe (12c)
-
3.4
Z-L-Ala-L-Phe-OMe (12d)
Z-L-Phe-L-Leu-OMe (12e)
Z-L-Phe-L-Ala-OMe (12f)
Z-L-Phe-L-Phe-OMe (12g)
Z-L-Tyr(Z)-L-Ala-OMe (12h)
Z-L-Tyr(Z)-L-Tyr-OMe (12i)
Fmoc-L-Phe-L-Phe-OMe (12j)
Fmoc-L-Ala-L-Leu-OMe (12k)
Fmoc-L-Ala-L-Ala-OMe (12l)
Fmoc-L-Ala-L-Phe-OMe (12m)
Fmoc-L-Pro-L-Ala-OMe (12n)
98.5/1.5
98.1/1.9
98.4/1.6
97.5/2.5
100/0
100/0
100/0
100/0
100/0
100/0
100/0
100/0
3.5
100/0
100/0
97.5/2.5
100/0
100/0
100/0
100/0
100/0
100/0
100/0
100/0
3.6
3.7
3.8
3.9
3.10
3.11
3.12
3.13
3.14
3.15
Fmoc-L-Asp(OtBu)-L-Ala-OMe
(12o)
3.16
3.17
3.18
3.19
3.20
3.21
Fmoc-L-Ser(tBu)-L-Ala-OMe (12p)
Fmoc-L-Thr(tBu)-L-Ala-OMe (12q)
Boc-Aib-L-Ala-OMe (12r)
88
75
74
71
68
89
96
95
100/0
100/0
-
98.8/1.2
98.8/1.2
100/0
100/0
99/1
99
Boc-L-Trp-L-Ala-OMe (12s)
Boc-L-Ala-L-Phe-OMe (12t)
Boc-Orn(Z)-Leu-OMe (12u)
85
*
99
100/0
100/0
100
100/0
a Enantiomeric purities were determined by GC analysis on a ChirasilVal column after degradation of peptides to amino acids and subsequent formation of
volatile N-Tfa-amino acid-OMe.
Moreover, all couplings proceeded with exceedingly low racemization (epimerization) of the
activated N-terminal amino acid residue. Only in the case of the C-terminal amino acid residue was
some racemization observed, particularly for the C-terminal phenylalanine residue. This may have
been caused by the harsh conditions of acidolytic degradation of the peptides to amino acids prior to
determination of their enantiomeric purity.
DMT/NMM/TsO- was also successfully applied in [2 + 1] fragment coupling (see Table 4) and [2 + 3]
fragment coupling (see Table 5).
This article is protected by copyright. All rights reserved.

Table 4. Tripeptides 13a-d synthesized in solution with DMT/NMM/TsO-using the [2+1] strategy.
Entry
Tripeptide
Yield [%]
Purity [%]
H2N-X1-X2-X3-COOH
%L / %D a
X1
X2
X3
4.1
4.2
4.3
Boc-L-Ala-Gly-L-Phe-OMe (13a)
Boc-L-Ala-Gly-L-Ala-OMe (13b)
78
81
84
90
91
92
100/0
100/0
100/0
-
-
98/2
100/0
100/0
Boc-L-Ala-L-Trp-L-Ala-OMe
*
(13c)
4.3
Boc-L-Ala-Aib-L-Leu-OMe (13d)
90
88
100/0
-
100/0
a Enantiomeric purities were determined by GC analysis on a ChirasilVal column after degradation of peptides to amino acids and subsequent formation of
volatile N-Tfa-amino acid-OMe.
In the case of tripeptide synthesis, the yield of the reaction ranged from 78-90% (Table 4). The
lowest yield was observed in the case of activation of the glycine residue (peptide 13a). This may
indicate that over-activation occurred, despite the progress of the formation of the superactive
triazine ester being controlled, resulting in the degradation of the reactive intermediates and thus a
lower reaction yield. The purity of the crude products in all cases was high, at around 90%.
Table 5. [2+3] strategy for synthesis of pentapeptides 14a-d in solution with DMT/NMM/TsO-.
Entry
Pentapeptide
Yield [%]
Purity [%]
H2N-X1-X2-X3-X4-X5-COOH
%L / %D a
X1
X2
X3
X4
X3
5.1
5.2
5.3
5.4
Z-L-Phe-L-Phe-L-Ala-Aib-L-Leu-OMe
75
71
76
70
99
95
89
98
99/1
99/1
99/1
-
100/0
(14a)
Z-L-Tyr(Z)-L-Tyr-L-Ala-L-Ala-L-Phe-OMe
99/1
98/2
99/1
97/3
99/1
100/0
98/2
99/1
100/
99/1
98/2
98/2
(14b)
Z-L-Ala-L-Phe-L-Ala–L-Trp-L-Ala-OMe
-
-
(14c)
Z-L-Tyr(Z)-L-Ala-L-Ala-Gly-L-Phe-OMe
100/0
(14d)
a Enantiomeric purities were determined by GC analysis on a ChirasilVal column after degradation of peptides to amino acids and subsequent formation of
volatile N-Tfa-amino acid-OMe.
Pentapeptides 14a-14d were obtained using the [2 + 3] strategy for fragment coupling, with
DMT/NMM/TsO- as the coupling reagent. The reaction yields were satisfactory, at above 70%, even
though in all cases the stoichiometry of the reagents used in the syntheses was maintained. The
purity of the raw products proved to be very high, in the range of 89-99%. The lowest purity was
observed for the pentapeptide Z-AlaPheAlaTrpAla-OMe (14c). However, the most important
This article is protected by copyright. All rights reserved.

evidence proving the high efficiency of DMT/NMM/TsO- for the coupling of fragments was the
optical purity of the final products. No loss of enantiomeric homogeneity was observed in any of the
synthesized peptides. As previously mentioned, the low content of amino acid D-enantiomers could
be the result of peptide racemization, occurring under the harsh conditions of acid hydrolysis (5 M
HCl, 20 h, 100°C) to free amino acids. Nevertheless, even in the case of the epimerization-sensitive
amino acid residue in position 2, L enantiomere content was in the range of 97-99%.
DMT/NMM/TsO- was furthermore used successfully in the synthesis of peptide derivatives
composed of unnatural amino acids. Reagent 7 was used in the synthesis of a peptide antiviral drug,
known by its commercial name as Boceprevir [25, 26] (Scheme 2). In the first stage, dipeptide 17 was
obtained, and in the second step precursor 20 of Boceprevir was obtained using the [2 + 1] fragment
coupling strategy.
Scheme 2. Synthesis of Boceprevir using DMT/NMM/TsO- (7) as a coupling reagent.
All synthetic steps necessary for the preparation of 20 were included within an optimized one-pot
procedure, without the isolation of superactive intermediate 2-acyloxy-4,6-dimethoxy-1,3,5-triazine.
The yield and purity of the synthesized products allowed Boceprevir to be obtained with a crude
product purity of 99.3%. It was thus possible to obtain pharmaceutical-grade purity after the
crystallization step.
Studies on the effectiveness of standard methods of peptide synthesis in the preparation of
Boceprevir intermediates showed that none of the tested approaches did not afforded the final
This article is protected by copyright. All rights reserved.

products with high purity. Data presented in table 6 show the results of the synthesis of dipeptide 17
and tripeptide 20 by using the active ester method (TBTU, EDC/HOBt), mixed anhydride methods
(isobutyl chloroformate), and acid chloride method (bis(trichloromethyl)carbonate).
Table 6. Synthesis of peptides 17 and 20 with classical coupling reagents.
Synthesis of dipeptide 17
Method of coupling
Purity of crude product
[%]a
Yield [%]
Purity after preparative HPLC [%]
Yield [%]
TBTU
66
80.5
91
75
72
85
93.6
-
63
-
EDC/HOBt
mixed anhydride method
isobutyl chloroformate
93.2
79
acid
chlorides
method
92
82
-
-
bis(trichloromethyl)carbonate (BTC)
Synthesis of tripeptide 20
Method of coupling
TBTU
Crude product
Products after preparative HPLC
Fr I - 38% - product
Fr I: purity = 99.3%, yield = 32%,
Fr II: purity = 99.8%, yield = 22%
Fr III: purity = 99.9%, yield = 15%
Fr II - 22% - depsipeptide
Fr III - 15% - dipeptide 17
EDC/HOBt
Fr I - 51% - product
Fr I: purity = 99.9 %, yield = 40%
Fr II: purity = 98.9%, yield = 18%
Fr II: purity = 99.6%,yield = 17%
Fr II - 23% - depsipeptide
Fr III - 26% - dipeptide 17
a HPLC 50-90%B in 20 min
In all cases, it was necessary to purify by preparative chromatography of both intermediates in order
to obtain satisfactory purity of the final products, which is technically disadvantageous as it reduces
the yield as well as increases the cost and time.
Conclusions
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TsO-),
an inexpensive and environmentally-friendly member of the of N-triazinylammonium family of
sulfonates, has been identified as a very effective coupling reagent for the synthesis of amides,
esters and peptides in solution. In this study, it was confirmed that DMT/NMM/TsO- can be used
with Z-, Fmoc- and Boc protected substrates as well as unnatural building blocks for the synthesis of
esters, thioesters, dipeptides and oligopeptides, with high yield, high crude product purity and low
levels of epimerization. Stoichiometric amounts of the reagents and standard synthetic procedures
were used, without any optimization stages. The results show that, in many cases, it is possible to
avoid time-consuming and costly chromatographic purification processes. DMT/NMM/TsO- was also
found to be very useful for the synthesis of oligopeptides. Using the fragment coupling strategy, final
peptides were obtained with high yields and very high purity, while most importantly preserving
enantiomeric homogeneity. This is particularly significant given that it is recommended to apply
This article is protected by copyright. All rights reserved.

additives when using classic coupling reagents, to suppress racemization. Thus, DMT/NMM/TsO- can
be considered as an effective tool for screening studies, allowing the preparation of diversified
products with satisfactory purity and yield. Moreover, after careful optimization even complex
products derived by coupling unusual building blocks can be obtained with high efficiency and to
pharmacological standards, while omitting chromatographic purification.
Experimental Section
General information
Thin-layer chromatography experiments were performed on silica gel (Merck; 60 Å F254). Spots
were visualized with UV light (254 and 366 nm) and with 1% ethanolic 4-(4-nitrobenzyl)pyridine
(NBP).
Melting points were determined using a Büchi apparatus, model 510.
Analytical RP-HPLC was performed with a Waters 600S HPLC system (Waters 2489 UV/Vis detector,
Waters 616 pump, Waters 717 plus autosampler, HPLC manager software from Chromax) using a
C18 column (25 cm x 4.6 mm, 5 mm; Sigma). HPLC was performed with a gradient of 0.1% TFA in H2O
(A) and 0.08% TFA in MeCN (B), at a flow rate of 1 mL/min with UV detection at 220 nm, Rt in min.
LC/MS spectra were recorded with a Waters ZQ 2000 Micromas or MS Bruker microOTOF-QIII.
IR spectra were recorded as KBr pellets or film with a Bruker ALPHA spectrometer or a PerkinElmer
Spectrum 100.
1H and 13C NMR spectra were recorded with a Bruker Avance DPX 250 (250 MHz) spectrometer, with
chemical shifts (ppm) relative to TMS used as an internal standard. Multiplicities are marked as s =
singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet.
Gas chromatography (GC) – Shimadzu GC-14A, FID (H2/air), split 1:50. Column: capilar Chirasil-Val
(25 m x 0,32 mm, thickness of film 0.2 µm, carrier gas: hel, pressure 0.45 atm. Temperature
program: 4 min in 90oC, next 4oC/min to 190oC and 3 min at 90oC.
Synthesis of 4-methoxy-N-(4-methylphenyl)benzamide (10a). Typical Procedure
4-Methoxy-benzoic acid (0.152 g, 1 mmol) and DIPEA (0.088 mL, 0.5 mmol) were added to a
vigorously stirred solution of DMT/NMM/TsO- (7) (0.413 g, 1 mmol) in CH2Cl2 (5 mL), cooled to 0oC.
Stirring was continued until the disappearance of condensing reagent 7 (TLC analysis, staining with
0.5% solution of NBP). Activation time: 40 min. After this time, p-tolylamine (0.107 g, 1 mmol) was
added and the mixture stirred for an additional 2h at 0oC. The mixture was left overnight at room
temperature. The mixture was diluted with CH2Cl2 (10 mL) and the solution was washed successively
with water, 0.5 M aqueous NaHSO4, water, 0.5 M aqueous NaHCO3 and with water again. The
organic layer was dried using MgSO4, filtered, and concentrated to dryness. The residue was dried
under a vacuum with P2O5 and KOH to a constant weight, affording 0.239 g 4-methoxy-N-p-tolyl-
benzamide (10a), yield = 99%, mp = 143-145oC, lit. [27] mp = 145-148oC.
This article is protected by copyright. All rights reserved.

Analysis: for C15H15NO2 (241.29): calculated: C 74.67%, H 6.27%, N 5.80%, O 13.26%, found: C
74.69%, H 6.25%, N 5.78%.
+
LC-MS: 242.68 ([M+H]+ C15H16NO2 , calc. 241.29).
Anal. RP-HPLC (3–97%B in 30 min): Rt = 22.81 min, purity 93%.
IR (film/NaCl): ν = 3339, 3031, 2963, 2862, 1651, 1596, 1514, 1500, 1402, 1308, 1237,
1101, 1031 [cm-1].
1H-NMR (250 MHz; CDCl3) δ = 2.30 (s, 3H, CH3); 3.84 (s, 3H, OCH3); 6.93 (d, 2H, J = 8.6 Hz, -C6H4-
OCH3); 7.13 (d, 2H, J = 8.1 Hz, -C6H4-CH3); 7.47 (d, 2H, J = 8.1 Hz, -C6H4-CH3); 7.81 (d, 2H, J= 8.6 Hz , -
C6H4-OCH3) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 21.3, 55.5, 113.6, 120.7, 129.6, 130.1, 132.5, 139.2, 160.1,
165.1 [ppm].
Synthesis of N-(4-methoxy-phenyl)-2,2-dimethyl-propionamide (10b)
Starting materials: 2,2-dimethyl-propionic acid (0.102 g, 1 mmol); 4-methoxy-phenylamine (0.123 g,
1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.088 mL, 0.5 mmol). Activation time: 45 min.
Product: N-(4-methoxy-phenyl)-2,2-dimethyl-propionamide: 0.191 g, yield = 92%, mp = 122-124˚C,
lit. [28] mp = 124-126°C.
Analysis: for C12H17NO2 (207.27): calculated: C 69.54%, H 8.27%, N 6.76%, O 15.44%, found: C
69.56%, H 8.26%, N 6.74%.
+
LC-MS: 208.68 ([M+H]+ C12H18NO2 , calc. 207.27).
Anal. RP-HPLC (3–97%B in 30 min): Rt = 19.87 min, purity 98%.
IR (film/NaCl): ν = 3303, 3130, 3009, 1644m 1622, 1602, 1579, 1542, 1509, 1459, 1312,
1246, 1170, 1033 [cm-1].
1H-NMR (250 MHz; CDCl3) δ = 1.09 (s, 9H, (CH3)3-); 3.71 (s, 3H, OCH3); 6.79 (d, 2H, J = 8.6 Hz, -C6H4-
OCH3); 7.24 (d, 2H, J = 8.2 Hz, -C6H4-NH) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 27.5, 39.8, 55.6, 114.8, 122.2, 137.1, 160.1, 172.1 [ppm].
Synthesis of N-Butyl-benzamide (10c)
Starting materials: benzoic acid (0.122 g, 1 mmol); butylamine (0.073 g, 1 mmol); DMT/NMM/TsO-
(0.413 g, 1 mmol); DIPEA (0.088 mL, 0.5 mmol). Activation time: 40 min. Product: N-Butyl-
benzamide: 0.159 g, yield = 90%, mp = 41-44°C, lit. [29] mp = 42-43oC.
Analysis: for C11H15NO (177.25): calculated: C 74.54%, H 8.53%, N 7.90%, O 9.03%, found: C 74.55%,
H 8.55%, N 7.92%.
LC-MS: 178.66 ([M+H]+ C11H16NO+, calc. 177.25).
Anal. RP-HPLC (3–97%B in 30 min): Rt = 19.56 min, purity 95%.
This article is protected by copyright. All rights reserved.

IR (film/NaCl): ν = 3314, 2958, 2931, 1635, 1578, 1536, 1490, 1465, 1365, 1304, 1223, 1151,
1074 [cm-1].
1H-NMR (250 MHz; CDCl3) δ = 0.87 (t, 3H, J = 6.5 Hz, CH3-CH2-); 1.37-1.49 (m, 4H, -CH2-CH2-);
3.36 (q, 2H, J = 4.8 Hz, -NH-CH2-); 7.47-7.51 (m, 3H, C6H5-); 7.82 (d, 2H, J = 6.9 Hz, C6H5-) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 13.5, 20.1, 31.4, 39.5, 127.5, 128.2, 130.1, 133.1, 167.2 [ppm].
Synthesis of N-butyl-2-phenyl-butyramide (10d)
Starting materials: 2-phenyl-butyric acid (0.164 g, 1 mmol); butylamine (0.073 g, 1 mmol);
DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.088 mL, 0.5 mmol). Activation time: 40 min. Product:
N-butyl-2-phenyl-butyramide: 0.204 g, yield = 93%, mp = 61-63oC.
Analysis: for C14H21NO (219.33): calculated: C 76.67%, H 9.65%, N 6.39%, O 7.29%, found: C 76.65%,
H 9.63%, N 7.27%.
LC-MS: 220.84 ([M+H]+ C14H22NO+, calc. 219.33).
Anal. RP-HPLC (3–97%B in 30 min): Rt = 21.17 min, purity 89%.
IR (film/NaCl): ν = 3298, 2960, 2931, 2873, 1641, 1544, 1495, 1454, 1365, 1273, 1227,
1189, 1115, 1031 [cm-1].
1H-NMR (250 MHz; CDCl3) δ = 0.81 (t, 3H, J = 6.4 Hz, CH3-CH2-); 0.86 (t, 3H, J = 6.6 Hz, CH3-CH2-);
1.22-1.29 (m, 4H, -CH2-CH2-); 1.52-1.72 (m, 2H, CH3-CH2-); 3.19 (q, 2H, J = 4.9 Hz, -NH-CH2-); 3.65 (dd,
1H, J1 = 6.5 Hz, J2 = 4.1 Hz, -CH-CH2-); 7.47-7.51 (m, 3H, C6H5-); 7.82 (d, 2H, J = 6.9 Hz, C6H5-) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 11.5, 13.8, 20.5, 26.4, 31.5, 39.6, 47.4, 127.5, 126.3, 128.9,
138.1, 172.4 [ppm].
Synthesis of (R)-3-methyl-N-p-tolyl-succinamic acid benzyl ester (10e)
Starting materials: Z-L-Ala-OH (0.223 g, 1 mmol), p-tolylamine (0.107 g, 1 mmol); DMT/NMM/TsO-
(0.413 g, 1 mmol); DIPEA (0.088 mL, 0.5 mmol). Activation time: 30 min. Product: (R)-3-methyl-N-p-
tolyl-succinamic acid benzyl ester: 0.275 g, yield = 88%, mp = 121-123oC.
Anal. RP-HPLC (3–97%B in 30 min): Rt = 16.22 min, purity 99%.
LC-MS: 313.87 ([M+H]+ C18H21N2O3 , calc. 312.37).
+
IR (film/NaCl): ν = 3302, 3278, 3123, 2971, 1686, 1654, 1597, 1562, 1451, 1323, 1243,
1202, 1105, 1071, 1027 [cm-1].
1H-NMR (250 MHz; CDCl3) δ = 1.43 (d, 3H, J = 7.5 Hz, CH3-CH-); 2.31 (s, 3H, CH3-C6H4-); 4.11 (q,
1H, J = 7.5 Hz, CH3-CH-); 5.08 (bs, 2H, -CH2O-); 7.13 (dd, 2H, J1 = 3.9 Hz, J2 = 7.3 Hz, C6H4-); 7.35-7.39
(m, 5H, C6H5-); 7.58 (dd, 2H, J1 = 3.9 Hz, J2 = 7.3 Hz, C6H4-) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 18.7, 21.6, 50.2, 66.5, 120.5, 127.8, 128.2, 128.9, 129.8 130.1,
137.6, 156.8, 168.9 [ppm].
This article is protected by copyright. All rights reserved.

Synthesis of allyl 4-methoxybenzoate (11a). Typical Procedure
4-Methoxy-benzoic acid (0.152 g, 1 mmol) and DIPEA (0.088 mL, 0.5 mmol) were added to a
vigorously stirred solution of DMT/NMM/TsO- (7) (0.413 g, 1 mmol) in CH2Cl2 (5 mL), cooled to 0°C.
Stirring was continued until the disappearance of condensing reagent 7 (TLC analysis, staining with
0.5% solution of NBP). Activation time: 40 min. After this time, allyl alcohol (0.058 g, 1 mmol) and
DIPEA (0.176 mL, 1 mmol) were added, and the mixture stirred for an additional 2h at 0oC. The
mixture was left overnight at room temperature. The mixture was diluted with CH2Cl2 (10 mL), and
the solution was washed successively with water, 0.5 M aqueous NaHSO4, water, 0.5 M aqueous
NaHCO3 and with water again. The organic layer was dried using MgSO4, filtered and concentrated to
dryness. The residue was dried under a vacuum with P2O5 and KOH to a constant weight, affording
0.158 g allyl 4-methoxybenzoate (11a), yield = 82%, bp = 128-133oC (p = 1.0 Torr), lit. [30] bp = 130-
140oC (p = 1.3 Torr). Anal. RP-HPLC (3-97%B in 30 min): Rt = 17.77 min, purity 99%.
LC-MS: 192.91 [M]+ C11H12O3 , calc. 192.22.
+
IR (film/NaCl): ν = 2928, 1692, 1548, 1560, 1536, 1500, 1464, 136, 1304, 1272, 1192, 1116,
788 [cm-1].
1H-NMR (250 MHz; CDCl3): δ = 3.88 (s, 3H, CH3O); 4.81 (d, 2H, J = 6 Hz, -O-CH2-CH=CH2); 5.24-5.43
(m, 2H, -CH2-CH=CH2); 5.98-6.09 (m, 1H, -CH2-CH=CH2); 6.91 (d, 2H, J = 8 Hz, arom.); 8.17 (d, 2H, J = 8
Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ: 55.5, 65.6, 113.9, 118.2, 123.0, 132.0, 132.7, 166.5 [ppm].
Synthesis of Fmoc-L-Ala-OAll (11b)
Starting materials: Fmoc-L-Ala-OH (0.311 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 40 min. Product: Fmoc-L-Ala-
OAll (11b): 0.302 g, yield = 86%, oil. Anal. RP-HPLC (20–97%B in 30 min): Rt = 6.18 min, purity 88%.
LC-MS: 352.88 ([M+H]+ C21H22NO4 , calc. 351.41).
+
IR (film/NaCl): ν = 3344, 2928, 1724, 1508, 1448, 1336, 1252, 1204, 1072, 764 [cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.41 (d, 3H, J = 10 Hz, CH3-CH-); 4.20 (t, 1H, J = 6.5 Hz, -CHCH2O);
4.39-4.46 (m, 3H, -CHCH2O + CH3-CH-); 4.66 (d, 2H, J = 6 Hz, -O-CH2-CH=CH2); 5.24-5.37 (m, 3H, -CH2-
CH=CH2 + -OCO-NH-); 5.93 (m, 1H, -CH2-CH=CH2); 7.31 (t, 2H, J = 7 Hz, arom.); 7.40 (t, 2H, J = 7 Hz,
arom.); 7.59 (d, 2H, J = 7 Hz, arom.); 7.76 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 18.6, 47.1, 49.5, 65.4, 66.8, 118.1, 119.8, 125.1, 127.1, 128.6,
132.1, 142.6, 160.1, 172.8 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.23 (L-Ala);
L/D = 100/0.
Synthesis of Fmoc-L-Phe-OAll (11c)
Starting materials: Fmoc-L-Phe-OH (0.387 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 45 min. Product: Fmoc-L-Phe-
This article is protected by copyright. All rights reserved.

OAll (11c): 0.372 g, yield = 87%, mp = 103-105oC, lit. [31] mp = 105-106oC. Anal. RP-HPLC (20–97%B
in 30 min): Rt = 8.33 min, purity 95%.
+
LC-MS: 428.88 ([M+H]+ C27H26NO4 , calc. 427.50).
IR (film/NaCl): ν = 3320, 3032, 2944, 1728, 1512, 1448, 1340, 1192, 1052, 760, 704 [cm-1].
1H-NMR (250 MHz; CDCl3): δ = 3.06 (broad d, 2H, J = 6.5 Hz, CH-CH2-C6H5); 4.17-4.23 (m, 1H, -
O-CH2-CH-); 4.30-4.44 (m, 2H, -O-CH2-CH-); 4.61 (d, 2H, J = 6 Hz, -O-CH2-CH=CH2); 4.72 (q, 1H, J = 6.5
Hz, -CH-CH2-C6H5); 5.23-5.34 (m, 3H, -CH2-CH=CH2 + -OCO-NH-); 5.86 (m, 1H, -CH2-CH=CH2); 7.09-7.77
(m, 13H, arom) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 38.3, 47.1, 54.7, 65.4, 66.8, 118.1, 120.1, 125.1, 127.2, 128.7, 129.1,
132.1, 136.4, 142.2, 156.1, 171.6 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 21.15 (D-Phe),
Rt = 21.45 (L-Phe), L/D = 99.5/0.5.
Synthesis of Fmoc-L-Glu(OtBu)-OAll (11d)
Starting materials: Fmoc-L-Glu(OtBu)-OH (0.426 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol);
DIPEA (0.264 mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 45 min. Product: Fmoc-
L-Glu(OtBu)-OAll (11d): 0.358 g, yield = 77%, mp = 67-69oC, lit. [32] mp = 69-70oC. Anal. RP-HPLC
(20–97%B in 30 min): Rt = 7.73 min, purity 93%.
+
LC-MS: 465.95 ([M+H]+ C27H32NO6 , calc. 465.55).
IR (film/NaCl): ν = 2976, 2928, 1724, 1512, 1448, 1368, 1332, 1152, 1056, 784 [cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.44 (s, 9H, (CH3)3CO-); 1.90-2.40 (m, 4H, -CH2-CH-); 4.22 (t, 1H, J =
7 Hz, -CH-CH2-O-); 4.30-4.45 (m, 2H, -CH-CH2-O-); 4.64 (d, 2H, J = 6 Hz, -OCH2CH=CH2); 5.20-5.40 (m,
2H, OCH2CH=CH2); 5.46 (m, 1H, NH); 5.89 (m, 1H, OCH2CH=CH2); 7.31 (t, 2H, J = 7 Hz, arom.); 7.40 (t,
2H, J = 7 Hz, arom.); 7.59 (d, 2H, J = 7 Hz, arom.); 7.76 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 28.0, 28.3, 31.6, 47.1, 53.8, 65.4, 66.9, 81.0, 118.9, 120.1, 125.2,
127.1, 128.7, 132.1, 142.2, 156.1, 171.1, 172.2 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 14.88 (L-Glu),
L/D = 100/0.
Synthesis of Fmoc-L-Ser(tBu)-OAll (11e)
Starting materials: Fmoc-L-Ser(tBu)-OH (0.383 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 35 min. Product: Fmoc-L-
Ser(tBu)-OAll (11e): 0.347 g, yield = 82%, mp = 59-62oC. lit. [33] mp = 58-60oC. Anal. RP-HPLC (20–
97%B in 30 min): Rt = 7.07 min, purity 93%.
+
LC-MS: 424.82 ([M+H]+ C25H30NO5 , calc. 423.51).
This article is protected by copyright. All rights reserved.

IR (film/NaCl): ν = 3448, 2976, 1724, 1508, 1448, 1392, 1364, 1336, 1192, 1104, 984, 784,
648 [cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.20 (s, 9H, -OC(CH3)3); 3.62 (dd, 1H, J1 = 9 Hz, J2 = 6 Hz, -CH-CH2-
O-); 3.87 (dd, 1H, J1 = 9 Hz, J2 = 6 Hz, -CH-CH2-O-); 4.23-4.29 (m, 1H, -CHCH2O-); 4.36-4.54 (m, 3H, -
CHCH2O- + -CHCH2OC(CH3)3); 4.68 (d, 2H, J = 7 Hz, -OCH2CH=CH2); 5.22-5.38 (m, 2H, OCH2CH=CH2);
5.62 (broad d, 1H, J = 4.5 Hz, NH); 5.66 (m, 1H, OCH2CH=CH2); 7.26 (t, 2H, J = 7 Hz, arom.); 7.37 (t, 2H,
J = 7 Hz, arom.); 7.60 (d, 2H, J = 7 Hz, arom.); 7.75 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 27.6, 47.1, 54.6, 65.4, 55.8, 69.4, 73.1, 118.1, 119.9, 125.0,
127.2, 128.6, 132.1, 142.2, 152.3, 171.7 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 10.32 (L-Ser),
Rt = 10.64 (D-Ser) L/D = 99.6/0.4.
Synthesis of Fmoc-L-Lys(Boc)-OAll (11f)
Starting materials: Fmoc-L-Lys(Boc)-OH (0.468 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 50 min. Product: Fmoc-L-
Lys(Boc)-OAll (11f): 0.488 g, yield = 96%, mp = 120-122oC, lit. [34] mp = 123oC. Anal. RP-HPLC (20–
97%B in 30 min): Rt = 8.25 min, purity 96%.
LC-MS: 509.92 ([M+H]+ C29H37N2O6 , calc. 508.62).
+
IR (film/NaCl): ν = 2936, 1720, 1504, 1448, 1368, 1248, 1168, 752 [cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.36-1.80 (m, 6H, -CH2-(CH2)3); 1.48 (s, 9H, (CH3)3COO); 3.10-3.12 (m,
2H, -NHCH2(CH2)3); 4.22-4.25 (m, 1H, -CHCH2O-); 4.38-4.42 (m, 3H, -CHCH2O- + -CH(CH2)4); 4.66 (d,
2H, J = 6 Hz, -OCH2CH=CH2); 5.42-5.39 (m, 3H, OCH2CH=CH2 + NHCO); 5.95 (m, 1H, OCH2CH=CH2);
7.26 (t, 2H, J = 7 Hz, arom.); 7.37 (t, 2H, J = 7 Hz, arom.); 7.60 (d, 2H, J = 7 Hz, arom.); 7.75 (d, 2H, J =
7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 22.1, 28.2, 29.4, 31.4, 40.6, 47.1, 53.5, 65.4, 66.9, 80.1, 118.1,
119.9, 125.1, 127.2, 128.6, 132.1, 142.2, 156.2, 171.6 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 11.43 (L-Lys),
Rt = 11.04 (D-Lys) L/D = 99.5/0.5.
Synthesis of Fmoc-L-Asp(OtBu)-OAll (11g)
Starting materials: Fmoc-L-Asp(OtBu)-OH (0.411 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol);
DIPEA (0.264 mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 40 min. Product: Fmoc-
L-Asp(OtBu)-OAll (11g): 0.388 g, yield = 86%, mp = 79-81oC, lit. [35] mp = 82-83oC. Anal. RP-HPLC (20-
97%B in 30 min): Rt = 7.06 min, purity 99%.
LC-MS: 453.12 ([M+H]+ C26H30NO6 , calc. 451.52).
+
IR (film/NaCl): ν = 3438, 2942, 1724, 1507, 1463, 1448, 1369, 1336 [cm-1].
This article is protected by copyright. All rights reserved.

1H-NMR (250 MHz; CDCl3): δ = 1.45 (s, 9H, (CH3)3CO-); 2.80, 2.95 (d AB, 2H, J1 = 16 Hz, J2 = 4 Hz, -
OCOCH2CH-); 4.20-4.55 (m, 4H, -CHCH2OCONH- + -OCOCH2CH-); 4.65 (d, 2H, J = 6 Hz, -OCH2CH=CH2);
5.20-5.40 (m, 2H, -OCH2CH=CH2); 5.81 (d, 1H, J = 7.5 Hz, NH); 5.93 (m, 1H, CH2-CH=CH2); 7.31 (t, 2H, J
= 7 Hz, arom.); 7.40 (t, 2H, J = 7 Hz, arom.); 7.59 (d, 2H, J = 7 Hz, arom.); 7.76 (d, 2H, J = 7 Hz, arom.)
[ppm].
13C-NMR (75 MHz, CDCl3): δ = 28.3, 31.6, 47.1, 53.8, 65.4, 66.9, 81.0, 118.9, 120.1, 125.2,
127.1, 128.7, 132.1, 142.2, 156.1, 171.1, 172.2 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min Rt = 14.13 (L-Asp),
L/D = 100/0.
Synthesis of Fmoc-L-Asp(OtBu)-OAll (11g) in the presence DCC
Starting materials: Fmoc-L-Asp(OtBu)-OH (0.411 g, 1 mmol), DCC (0.206 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 40 min. Product: Fmoc-L-Asp(OtBu)-
OAll (11g): 0.316 g, yield = 70%, anal. RP-HPLC (20–97%B in 30 min): Rt = 7.04 min, purity 92%.
Product identical to that described above.
Synthesis of Fmoc-L-Asp(OtBu)-OAll (11g) in the presence TBTU
Starting materials: Fmoc-L-Asp(OtBu)-OH (0.411 g, 1 mmol), TBTU (0.321 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 40 min. Product: Fmoc-L-Asp(OtBu)-
OAll (11g): 0.316 g, yield = 70%, anal. RP-HPLC (20–97%B in 30 min): Rt = 7.05 min, purity 99%.
Product identical to that described above.
Synthesis of Fmoc-L-Asp(OtBu)-OAll (11g) in the presence HATU
Starting materials: Fmoc-L-Asp(OtBu)-OH (0.411 g, 1 mmol), HATU (0.380 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 40 min. Product: Fmoc-L-Asp(OtBu)-
OAll (11g): 0.325 g, yield = 72%, anal. RP-HPLC (20–97%B in 30 min): Rt = 7.06 min, purity 98%.
Product identical to that described above.
Synthesis of Fmoc-L-Asp(OtBu)-OAll (11g) in the presence BOP
Starting materials: Fmoc-L-Asp(OtBu)-OH (0.411 g, 1 mmol), BOP (0.442 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol), allyl alcohol (0.058 g, 1 mmol). Activation time: 40 min. Product: Fmoc-L-Asp(OtBu)-
OAll (11g): 0.266 g, yield = 59%, anal. RP-HPLC (20–97%B in 30 min): Rt = 7.05 min, purity 99%.
Product identical to that described above.
Synthesis of Fmoc-L-Asp(OtBu)-OPfp (11h)
Starting materials: Fmoc-L-Asp(OtBu)-OH (0.411 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol);
DIPEA (0.264 mL, 1.5 mmol), pentafluorophenol (0.184 g, 1 mmol). Activation time: 40 min. Product:
20
Fmoc-L-Asp(OtBu)-OPfp (11h): 0.508 g, yield = 88%, mp. = 93-95oC, lit. [36] mp. = 94.6-95.8oC, [α]D
This article is protected by copyright. All rights reserved.

= -2.4 (c = 1.0, CHCl3), lit. [37] α]D20 = -2.5 (c = 1.0, CHCl3). Anal. RP-HPLC (20–97%B in 30 min): Rt =
8.77 min, purity 93%.
+
LC-MS: 578.91 ([M+H]+ C29H25F5NO6 , calc. 577.51).
IR (film/NaCl): ν = 3320, 3027, 2966, 1738, 1513, 1441, 1347, 1244, 1190, 1050, 767, 709
[cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.38 (s, 9H); 2.97 (d, 2H, J = 6.2 Hz); 4.38 (d, 2H, J = 3.1 Hz); 4.49 (t,
1H, J = 3.1 Hz); 5.06 (t, 1H, J = 6.2 Hz); 5.53 (d, 1H, J = 7.0 Hz, OCO-NH-); 7.29 (t, 2H, J = 7.2 Hz,
arom.); 7.39 (t, 2H, J = 7.2 Hz, arom.); 7.58 (d, 2H, J = 7.2 Hz, arom.); 7.75 (d, 2H, J = 7.2 Hz, arom.)
[ppm].
13C-NMR (75 MHz, CDCl3): δ = 28.0, 36.1, 47.1, 53.8, 66.9, 119.9, 125.1, 127.2, 128.6, 138.3,
139.1, 141.0, 142.2, 156.1, 171.5, 172.3 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min Rt = 14.13 (L-Asp),
L/D = 100/0.
Synthesis of Fmoc-L-Ala-OPfp (11i)
Starting materials: Fmoc-L-Ala-OH (0.311 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), pentafluorophenol (0.184 g, 1 mmol). Activation time: 30 min. Product: Fmoc-
L-Ala-OPfp (11i): 0.396 g, yield = 83%, mp = ,172-174oC, lit. [38] mp = 171-173oC, [α]D20 = -22.5 (c =
1.0, CHCl3), lit. [39] [α]D20 = -22.2 (c = 1.0, CHCl3). Anal. RP-HPLC (20–97%B in 30 min): Rt = 7.01 min,
purity 88%.
LC-MS: 478.89 ([M+H]+ C24H17F5NO4 , calc. 477.39).
+
IR (film/NaCl): ν = 3322, 3030, 2965, 1742, 1510, 1440, 1340, 1245, 1192, 1050, 760, 704 [cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.44 (d, 3H, J = 7.4 Hz); 4.38 (d, 2H, J = 3.1 Hz); 4.49 (t, 1H, J = 3.1 Hz);
4.62 (q, 1H, J = 7.4 Hz); 5.53 (d, 1H, J = 7.0 Hz, OCO-NH-); 7.29 (t, 2H, J = 7.2 Hz, arom.); 7.39 (t, 2H, J
= 7.2 Hz, arom.); 7.58 (d, 2H, J = 7.2 Hz, arom.); 7.75 (d, 2H, J = 7.2 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 18.6, 49.5, 47.1, 66.9, 119.9, 125.1, 127.2, 128.8, 138.3, 139.1,
141.2, 155.9, 171.5 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.23 (L-Ala);
L/D = 100/0.
Synthesis of Fmoc-L-Ala-ONSu (11j)
Starting materials: Fmoc-L-Ala-OH (0.311 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), N-hydroxysusuccinimide (0.115 g, 1 mmol). Activation time: 30 min. Product:
Fmoc-L-Ala-ONSu (11j): 0.319 g, yield = 78%, oil, [α]D20 = -23.1 (c = 1.0, CHCl3), lit. [40] [α]D20 = -23
(c = 1.0, CHCl3). Anal. RP-HPLC (20–97%B in 30 min): Rt = 6.81 min, purity 95%.
+
LC-MS: 409.91 ([M+H]+ C22H21N2O6 , calc. 408.41).
This article is protected by copyright. All rights reserved.

IR (film/NaCl): ν = 3320, 3035, 2960, 1748, 1501, 1440, 1346, 1248, 1188, 1052, 761, 705
[cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.44 (d, 3H, J = 7.4 Hz); 2.83 (ddd, 4H, J = 15.0 Hz, J = 8.1 Hz, J = 4.3
Hz); 4.38 (d, 2H, J = 3.1 Hz); 4.49 (t, 1H, J = 3.1 Hz); 4.62 (q, 1H, J = 7.4 Hz); 5.53 (d, 1H, J = 7.0 Hz,
OCO-NH-); 7.29 (t, 2H, J = 7.2 Hz, arom.); 7.39 (t, 2H, J = 7.2 Hz, arom.); 7.58 (d, 2H, J = 7.2 Hz,
arom.); 7.75 (d, 2H, J = 7.2 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 18.1, 25.6, 47.1, 50.3, 66.9, 119.9, 125.1, 127.2, 128.6, 142.2,
155.9, 169.1, 169.7 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.23 (L-Ala);
L/D = 100/0.
Synthesis of Fmoc-L-Ala-SPh (11k)
Starting materials: Fmoc-L-Ala-OH (0.311 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), thiophenol (0.110 g, 1 mmol). Activation time: 30 min. Product: Fmoc-L-Ala-
SPh (11k): 0.383 g, yield = 94%, oil. Anal. RP-HPLC (20–97%B in 30 min): Rt = 7.21 min, purity 97%.
LC-MS: 404.89 ([M+H]+ C24H22NO3S+, calc. 403.50).
IR (film/NaCl): ν = 3321, 3030, 2965, 1752, 1510, 1440, 1340, 1245, 1192, 1050, 771, 714
[cm-1].
1H-NMR (250 MHz; CDCl3): δ = 1.44 (d, 3H, J = 7.4 Hz); 4.38 (d, 2H, J = 3.1 Hz); 4.49 (t, 1H, J = 3.1 Hz);
4.62 (q, 1H, J = 7.4 Hz); 5.53 (d, 1H, J = 7.0 Hz, OCO-NH-); 7.17 (t, 1H, J = 7.5 Hz, arom); 7.24 (dd, 2H, J
= 7.5 Hz, J = 7.9 Hz, arom); 7.29 (t, 2H, J = 7.2 Hz, arom.); 7.39 (t, 2H, J = 7.2 Hz, arom.); 7.48 (d, 2H, J
= 7.8 Hz, arom); 7.58 (d, 2H, J = 7.2 Hz, arom.); 7.75 (d, 2H, J = 7.2 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 20.3, 47.2, 58.7, 66.9, 119.9, 125.1, 127.4, 128.6, 129.1, 131.4,
134.8, 142.2, 155.5, 193.4 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.23 (L-Ala);
L/D = 100/0.
Synthesis of Fmoc-L-Ser(tBu)-SPh (11l)
Starting materials: Fmoc-L-Ser(tBu)-OH (0.383 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), thiophenol (0.110 g, 1 mmol). Activation time: 45 min. Product: Fmoc-L-
Ser(tBu)-SPh (11l): 0.428 g, yield = 90%, oil, [α]D20 = -25.7 (c = 1.0, CHCl3), lit. [41] [α]D20 = -23.8 (c =
0.8, CHCl3). Anal. RP-HPLC (20–97%B in 30 min): Rt = 7.96 min, purity 96%.
LC-MS: 477.03 ([M+H]+ C28H30NO4S+, calc. 475.61).
IR (film/NaCl): ν = 3323, 3031, 2960, 1744, 1502, 1449, 1340, 1243, 1188, 1057, 760, 710
[cm-1].
This article is protected by copyright. All rights reserved.

1H-NMR (250 MHz; CDCl3): δ = 1.29 (9H, s); 4.38 (d, 2H, J = 3.1 Hz); 4.49 (t, 1H, J = 3.1 Hz); 4.68 (d,
2H, J = 6.8 Hz); 4.71 (t, 1H, J = 6.8 Hz); 5.53 (d, 1H, J = 7.0 Hz, OCO-NH-); 7.17 (t, 1H, J = 7.5 Hz, arom);
7.24 (dd, 2H, J = 7.5 Hz, J = 7.9 Hz, arom); 7.29 (t, 2H, J = 7.2 Hz, arom.); 7.39 (t, 2H, J = 7.2 Hz, arom.);
7.48 (d, 2H, J = 7.8 Hz, arom); 7.58 (d, 2H, J = 7.2 Hz, arom.); 7.75 (d, 2H, J = 7.2 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 27.6, 47.2, 58.1, 66.9, 69.3, 73.5, 119.9, 125.1, 127.1, 128.6,
129.1, 131.4, 134.6, 142.2, 155.5, 193.4 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 10.32 (L-Ser),
Rt = 10.64 (D-Ser) L/D = 99/1.
Synthesis of Fmoc-L-Ala-S(CH2)3CH3 (11m)
Starting materials: Fmoc-L-Ala-OH (0.311 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), butanethiol (0.091 g, 1 mmol). Activation time: 30 min. Product: Fmoc-L-Ala-
S(CH2)3CH3 (11m): 0.337 g, yield = 88%, oil. Anal. RP-HPLC (20–97%B in 30 min): Rt = 7.31 min, purity
95%.
LC-MS: 384.95 ([M+H]+ C22H26NO3S+, calc. 383.51).
IR (film/NaCl): ν = 3320, 3037, 2960, 1750, 1510, 1449, 1345, 1245, 1192, 1050, 762, 709
[cm-1].
1H-NMR (250 MHz; CDCl3): δ = 0.89 (3H, t, J = 6.5 Hz); 1.29-1.33 (m, 4H); 1.44 (d, 3H, J = 7.4 Hz); 3.35
(2H, t, J = 7.2 Hz); 4.38 (d, 2H, J = 3.1 Hz); 4.49 (t, 1H, J = 3.1 Hz); 4.62 (q, 1H, J = 7.4 Hz); 5.53 (d, 1H, J
= 7.0 Hz, OCO-NH-); 7.29 (t, 2H, J = 7.2 Hz, arom.); 7.39 (t, 2H, J = 7.2 Hz, arom.); 7.58 (d, 2H, J = 7.2
Hz, arom.); 7.75 (d, 2H, J = 7.2 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 13.7, 20.3, 21.9, 30.1, 31.2, 47.1, 58.1, 66.9, 119.9, 125.1,
127.2, 128.6, 142.2, 155.5, 193.4 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.20 (D-Ala), Rt
= 5.23 (L-Ala), L/D = 99/1.
Synthesis of Fmoc-L-Ser(tBu)-S(CH2)3CH3 (11n)
Starting materials: Fmoc-L-Ser(tBu)-OH (0.383 g, 1 mmol), DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol), butanethiol (0.091 g, 1 mmol). Activation time: 45 min. Product: Fmoc-L-
Ser(tBu)-S(CH2)3CH3 (11n): 0.364 g, yield = 80%, oil. Anal. RP-HPLC (20-97%B in 30 min): Rt = 8.11
min, purity 97%.
LC-MS: 457.12 ([M+H]+ C26H34NO4S+, calc. 455.62).
IR (film/NaCl): ν = 3322, 3031, 2955, 1748, 1510, 1441, 1340, 1241, 1190, 1050, 778, 702
[cm-1].
1H-NMR (250 MHz; CDCl3): δ = 0.89 (3H, t, J = 6.5 Hz); 1.29 (13H, s); 1.29-1.33 (m, 4H); 3.35 (2H, t, J =
7.2 Hz); 3.87 (2H d, J = 6.8 Hz), 4.38 (d, 2H, J = 3.1 Hz); 4.49 (t, 1H, J = 3.1 Hz); 4.62 (1H, dt, J = 3.10
This article is protected by copyright. All rights reserved.

Hz, J = 6.85 Hz); 5.53 (d, 1H, J = 7.0 Hz, OCO-NH-); 7.29 (t, 2H, J = 7.2 Hz, arom.); 7.39 (t, 2H, J = 7.2
Hz, arom.); 7.58 (d, 2H, J = 7.2 Hz, arom.); 7.75 (d, 2H, J = 7.2 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 13.3, 21.9, 27.6, 30.2, 31.1, 47.2, 58.1, 66.9, 69.3, 73.5, 119.9,
125.1, 127.3, 128.7, 142.2, 155.5, 193.4 [ppm].
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 10.32 (L-Ser),
Rt = 10.64 (D-Ser) L/D = 98/2.
Synthesis of Z-Aib-Ala-OMe (12a): Typical Procedure
Z-Aib-OH (0.237 g, 1 mmol) and DIPEA (0.088 mL, 0.5 mmol) were added to a vigorously stirred
solution of DMT/NMM/TsO- (0.413 g, 1 mmol) in CH2Cl2 (5 mL), cooled to 0oC. The stirring was
continued until the disappearance of a condensing reagent 5 (TLC analysis, staining with 0.5%
solution of NBP), activation time = 1 h. After which time, HCl*L-Ala-OMe (0.139 g, 1 mmol) and
DIPEA (0.176 mL, 1 mmol) were added and the mixture stirred for an additional 2 h at 0oC. The
mixture was left overnight at room temperature. The mixture was diluted with CH2Cl2 (10 mL) and
the solution was washed successively with water, 0.5 M aqueous NaHSO4, water, 0.5 M aqueous
NaHCO3 and with water again. The organic layer was dried with MgSO4, filtered and concentrated to
dryness. The residue was dried under a vacuum with P2O5 and KOH to a constant weight, affording 0.
277 g Z-Aib-Ala-OMe, yield = 85%, mp = 67-68oC, lit. [42] mp = 68.6-69.6oC. Anal. RP-HPLC (50-100%B
in 30 min): Rt = 5.77 min, purity 95%.
IR (film/NaCl): ν = 3678, 3608, 3430, 3017, 2965, 2398, 1734, 1672, 1512, 1451, 1420, 1250, 1224,
1191, 1170, 1151, 1086, 1066, 1020 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.36 (d, 3H, J = 7.1 Hz, CH3-CH-); 1.53, 1.54 (s, 3H + 3H, (CH3)2C-); 3.74
(s, 3H, CH3O-); 4.46 (qu, 1H, J = 7.1 Hz, CH3CH-); 5.10 (s, 2H, -CH2O-); 5.34 (s, 1H, NH-COO), 6.80
(broad d, 1H, J = 7 Hz, NH); 7.28-7.36 (s, 5H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 25.1, 48.1, 52.3, 58.1, 66.5, 127.9, 128.5, 128.9, 155.6,
172.9, 175,6 [ppm].
+
LC/MS: 323.3 ([M+H]+, C16H22N2O5 ; calc. 322.36).
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.23 (L-Ala);
L/D = 100/0; Rt = 6.88 (Aib).
Synthesis of Z-Aib-Phe-OMe (12b)
Starting materials: Z-Aib-OH (0.237 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264 mL, 1.5 mmol) and H-Phe-OMe*HCl
(0.216 g, 1 mmol). Product: Z-Aib-Phe-OMe (0.323 g, 81%), mp = 96-97oC, lit. [42] mp = 94.5-95.5oC. Anal. RP-HPLC (50–100%B in 30 min):
Rt = 6.52 min, purity 99%.
IR (film/NaCl): ν = 3422, 3019, 2950, 1731, 1672, 1498, 1452, 1362, 1258 [cm-1].
This article is protected by copyright. All rights reserved.

1H-NMR (250 MHz, CDCl3): δ = 1.47 (s, 6H, (CH3)2C-); 3.08 (d, 2H, J = 7 Hz, -CH2CH-); 3.71 (s, 3H, CH3O-
); 4.84 (q, 1H, J = 7 Hz, -CH2CH-); 5.07 (s, 2H, -CH2O-); 5.27 (s, 1H, NH-COO); 6.68 (d, 1H, J = 7 Hz,
CONH), 7.06-7.82 (m,10H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 25.1, 37.6, 52.3, 53.5, 58.1, 66.5, 127.8, 128.1, 128.5, 128.9,
129.1, 136.3, 155.6, 171.3, 175.6, [ppm].
LC/MS: 399.5 ([M+H]+, C22H26N2O5 ; calc. 398.5).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 6.78 (Aib); Rt =
21.45 (L-Phe); L/D = 100/0.
Synthesis of Z-Aib-Leu-OMe (12c)
Starting materials: Z-Aib-OH (0.237 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264 mL,
1.5 mmol) and H-Leu-OMe*HCl (0.182 g, 1 mmol). Product: Z-Aib-Leu-OMe (0.306 g, 84%), mp = 78-
80oC, lit. [43] mp = 77-80oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 6.40 min, purity 99%.
IR (film/NaCl): ν = 3684, 3620, 3433, 2975, 2895, 2400, 1735, 1717, 1680, 1559, 1512, 1417
[cm-1].
1H-NMR (250 MHz, CDCl3): δ = 0.91 (d, 6H, J = 6 Hz, (CH3)2-CH-); 1.45-1.7 (m, 3H, -CH2CH-); 1.53,
1.55 (s + s, 3H + 3H, (CH3)2C-); 3.71 (s, 3H, CH3O-); 4.59 (dt, 1H, J1 = 8.1 Hz, J2 = 4.5 Hz, -CH2CH-); 5.09
(s, 2H, -CH2O-); 5.31 (s, 1H, NHCOO); 6.61 (d, 1H, J = 5 Hz, NH); 7.35 (s, 5H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 22.6, 24.6, 25.1, 38.2, 52.3, 53.8, 58.1, 66.5, 127.9, 128.5,
128.9, 155.6, 172.8, 175.6 [ppm].
LC/MS: 365.4 ([M+H]+, C19H28N2O5 ; calc. 364.5).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 6.49 (Aib); Rt =
10.65 (L-Leu); L/D = 100/0.
Synthesis of Z-Ala-Phe-OMe (12d)
Starting materials: Z-Ala-OH (0.223 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264 mL,
1.5 mmol) and H-Phe-OMe*HCl (0.216 g, 1 mmol). Product: Z-Ala-Phe-OMe (0.284 g, 74%), mp =
102-103oC, lit. [44] mp = 104-106oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 7.11 min, purity 80%.
IR (film/NaCl): ν = 3306, 3088, 3064, 2951, 1719, 1660, 1584, 1521, 1497, 1364, 1212, 1178, 1114,
1068 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.44 (d, 3H, J = 6.2 Hz, CH3-CH-); 3.19 (dd, 2H, J1 = 4.1 Hz, J2 = 7.2
Hz, -CH2-CH-); 3.65 (s, 3H, CH3O-); 4.31 (q, 1H, J = 6.2 Hz, CH3-CH-); 4.75 (q, 1H, J = 6.9 Hz, -CH2-CH-);
5.04 (s, 2H, -CH2-O-); 7.27-7.36 (m, 10H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.5, 37.6, 50.5, 52.3, 53.2, 66.5, 127.9, 128.3, 128.6, 128.9,
129.1, 136.3, 154.4, 171.3, 172.2 [ppm].
This article is protected by copyright. All rights reserved.

LC/MS: 385.5 ([M+H]+, C21H24N2O5 ; calc. 384.4).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.20 (D-Ala), Rt
= 5.23 (L-Ala), L/D = 98.5/1.5; Rt = 21.15 (D-Phe), Rt = 21.45 (L-Phe), L/D = 99/1.
Synthesis of Z-Phe-Leu-OMe (12e)
Starting materials: Z-Phe-OH (0.299 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Leu-OMe*HCl (0.182 g, 1 mmol). Product: Z-Phe-Leu-OMe (0.303 g, 71%), mp =
109-111oC, lit. [45] mp = 110-112C. Anal. RP-HPLC (50-100%B in 30 min): Rt = 7.95 min, purity 90%.
IR (film/NaCl): ν = 3377, 3080, 2956, 2868, 1746, 1691, 1656, 1541, 1454, 1434, 1368,
1260, 1222, 1147, 1055 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 0.90 (d, 6H, J = 6.1 Hz, (CH3)2-CH-); 1.44-1.72 (m, 3H, -CH2CH-); 3.20
(dd, 2H, J1 = 4.1 Hz, J2 = 7.2 Hz, -CH2-CH-); 3.62 (s, 3H, CH3O-); 4.51 (dt, 1H, J1 = 8.1 Hz, J2 = 4.5 Hz, -
CH2CH-); 4.77 (q, 1H, J = 6.9 Hz, -CH2-CH-); 7.25-7.41 (m, 10H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 22.6, 24.6, 38.2, 38.6, 52.3, 53.8, 55.8, 66.5, 127.9, 128.5,
128.7, 128.9, 129.1, 137.1, 155.8, 170.9, 172.8 [ppm].
LC/MS: 427.5 ([M+H]+, C24H30N2O5 ; calc. 426.5).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 10.65 (L-Leu),
L/D = 100/0; Rt = 21.16 (D-Phe), Rt = 21.44 (L-Phe), L/D = 98.1/1.9.
Synthesis of Z-Phe-Ala-OMe (12f)
Starting materials: Z-Phe-OH (0.299 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Z-Phe-Ala-OMe (0.296 g, 77%), mp =
122-124oC, lit. [46] mp = 125-126oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 6.98 min, purity 89%.
IR (film/NaCl): ν = 3294, 3031, 2950, 1735, 1690, 1645, 1530, 1454, 1371, 1284, 1236,
1216, 1140, 1045 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.44 (d, 3H, J = 6.1 Hz, CH3-CH-); 3.21 (dd, 2H, J1 = 4.1 Hz, J2 = 7.4
Hz, -CH2-CH-); 3.64 (s, 3H, CH3O-); 4.32 (q, 1H, J = 6.1 Hz, CH3-CH-); 4.75 (q, 1H, J = 7.1 Hz, -CH2-CH-);
5.04 (s, 2H, -CH2-O-); 7.26-7.39 (m, 10H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 38.4, 47.8, 52.3, 55.8, 66.5, 129.2, 127.9, 128.5, 128.7,
128.9, 137.1, 155.8, 171.2, 172.9 [ppm].
LC/MS: 385.4 ([M+H]+, C21H24N2O5+; calc. 384.4).
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.23 (L-Ala),
L/D = 100/0; Rt = 21.14 (D-Phe), Rt = 21.45 (L-Phe), L/D = 98.4/1.6.
This article is protected by copyright. All rights reserved.

Synthesis of Z-Phe-Phe-OMe (12g)
Starting materials: Z-Phe-OH (0.299 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Phe-OMe*HCl (0.216 g, 1 mmol). Product: Z-Phe-Phe-OMe (0.341 g, 74%), mp
= 133-136oC, lit. [47] mp = 136-138oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 8.12 min, purity
93%.
IR (film/NaCl): ν = 3304, 3274, 3064, 2961, 1733, 1686, 1652, 1535, 1494, 1453, 1384,
1286, 1258, 1197, 1083 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 2.89-3.19 (m, 4H, (-CH2-CH-)2); 3.55 (s, 3H, CH3O-); 4.81-4.88 (m, 2H, (-
CH2-CH-)2); 5.03 (s, 2H, -CH2-O-); 7.06-7.77 (m, 15H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 37.6, 38.4, 52.4, 53.2, 55.8, 66.5, 127.9, 128.5, 128.7, 128.9, 129.1,
137.1, 155.8, 170.9, 171.3 [ppm].
LC/MS: 461.6 ([M+H]+, C27H28N2O5 ; calc. 460.5).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 21.15 (D-Phe),
Rt = 21.44 (L-Phe), L/D = 97.5/2.5.
Synthesis of Z-Tyr(Z)-Ala-OMe (12h)
Starting materials: Z-Tyr(Z)-OH (0.449 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Z-Tyr(Z)-Ala-OMe (0.376 g, 70%), mp
= 178-180oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 9.02 min, purity 94%.
IR (film/NaCl): ν = 3302, 3065, 3035, 2951, 1748, 1656, 1508, 1449, 1374, 1204, 1048, 1033
[cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.22 (d, 3H, J = 6.4 Hz, CH3-CH-); 2.16 (dd, 2H, J1 = 4.6 Hz, J2 = 8.4
Hz, -CH2-CH-); 3.52 (s, 3H, CH3O-); 4.11 (q, 1H, J = 6.4 Hz, CH3-CH-); 4.53-4.60 (m, 1H, -CH2-CH-); 5.05
(s, 2H, -CH2-O-); 5.18 (s, 2H, -CH2-O-); 7.16-7.55 (m, 14H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 38.5, 47.8, 52.3, 55.8, 66.5, 70.3, 114.6, 127.9, 128.5,
128.9, 129.8, 153.1, 155.8, 171.2, 172.9 [ppm].
LC/MS: 535.5 ([M+H]+, C29H30N2O8 ; calc. 534.6).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.22 (L-Ala),
L/D = 100/0; Rt = 23.55 (L-Tyr), L/D = 100/0.
Synthesis of Z-Tyr(Z)-Tyr-OMe (12i)
Starting materials: Z-Tyr(Z)-OH (0.449 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Tyr-OMe*HCl (0.232 g, 1 mmol). Product: Z-Tyr(Z)-Tyr-OMe (0.457 g, 73%), mp
= 182-185oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 9.16 min, purity 86%.
IR (film/NaCl): ν = 3303, 3033, 2953, 1748, 1659, 1589, 1508, 1446, 1371, 1204, 1110,
1048, 1019 [cm-1].
This article is protected by copyright. All rights reserved.

1H-NMR (250 MHz, CDCl3): δ = 2.19 (dd, 2H, J1 = 4.6 Hz, J2 = 8.4 Hz, -CH2-CH-); 3.08 (dd, 2H, J1 = 4.6,
J2 = 8.0 Hz, -CH2-CH-); 3.55 (s, 3H, CH3O-); 4.53-4.60 (m, 1H, -CH2-CH-); 4.80-4.91 (m, 1H, -CH2-CH-);
5.05 (s, 2H, -CH2-O-); 5.18 (s, 2H, -CH2-O-); 6.32 (d, 2H, J = 7.1 Hz, C6H4-); 6.97 (d, 2H, J = 7.2 Hz, C6H4-
); 7.33-7.55 (m, 14H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 37.6, 38.4, 52.5, 53.2, 55.8, 66.5, 70.3, 115.7, 116.6, 127.9,
128.5, 128.9, 129.8, 130.5, 153.1, 155.8, 170.9, 171.3 [ppm].
LC/MS: 627.7 ([M+H]+, C35H34N2O9 ; calc. 626.7).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 23.55 (L-Tyr),
L/D = 100/0.
Synthesis of Fmoc-Phe-Phe-OMe (12j)
Starting materials: Fmoc-Phe-OH (0.387 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Phe-OMe*HCl (0.216 g, 1 mmol). Product: Fmoc-Phe-Phe-OMe (0.477 g,
87%), mp = 169-171oC. Anal. RP-HPLC (50-100%B in 30 min): Rt = 11.54 min, purity 84%.
IR (film/NaCl): ν = 3422, 1729, 1680, 1494, 1452, 1401, 1320 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 2.96 broad d, 2H, J = 6.5 Hz, -CH-CH2-C6H5); 3.08 (broad d, 2H, J
= 6.5 Hz, -CH-CH2-C6H5); 3.55 (s, 3H, CH3O-); 4.13-4.22 (m, 1H, -O-CH2-CH-); 4.25-4.45 (m, 4H, -O-CH2-
CH- + (-CH-CH2-C6H5)2); 5.38 (broad d, 1H, J = 5 Hz,
OCONH); 7.15-7.35 (m, 13H, arom. + NH), 7,43 (t, 2H, J = 7.5 Hz, arom.); 7.52 (t, 2H, J = 6.5 Hz,
arom.); 7.76 (d, 2H, J = 7.5 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 37.6, 38.4, 47.1, 52.3, 53.2, 55.8, 66.9, 119.9, 125.1, 127.1,
128.4, 128.7, 120.9, 129.1, 142.2, 155.5, 170.9, 171.3 [ppm].
LC/MS: 549.7 ([M+H]+, C34H32N2O5 ; calc. 548.6).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 21.45 (L-Phe);
L/D = 100/0.
Synthesis of Fmoc-Ala-Leu-OMe (12k)
Starting materials: Fmoc-Ala-OH (0.329 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Leu-OMe*HCl (0.182 g, 1 mmol). Product: Fmoc-Ala-Leu-OMe (0.381 g, 87%),
mp = 123-125oC, lit. [48] mp = 124-126oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 10.92 min,
purity 96%.
IR (film/NaCl): ν = 3608, 3422, 3017, 2973, 1739, 1711, 1676, 1506, 1448 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 0.88 (d, 6H, J = 4 Hz, (CH3)2-CH-); 1.39 (d, 3H, J = 7 Hz, CH3CH-);
1.50-2.05 (m, 3H, (CH3)2CHCH2-); 3.71 (s, 3H, CH3O-); 4.20 (t, 1H, J = 3 Hz, -CHCH2O-); 4.26-4.40 (m,
1H, CH-NH); 4.38 (d, 2H, J = 3 Hz, -CHCH2O); 4.59 (m, 1H, CHCH2CH); 5.53 (d, 1H, J = 7 Hz, OCO-NH-);
This article is protected by copyright. All rights reserved.

6.56 (d, 1H, J = 7 Hz, CO-NH-); 7.29 (t, 2H, J = 7 Hz, arom.); 7.39 (t, 2H, J = 7 Hz, arom.); 7.58 (d, 2H, J
= 7 Hz, arom.); 7.75 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.5, 22.4, 24.4, 38.2, 47.1, 50.5, 52.4, 53.8, 66.9, 119.9, 125.1,
127.1, 128.6, 142.2, 155.9, 172.2, 172.8 [ppm].
LC/MS: 439.6 ([M+H]+, C25H30N2O5 ; calc. 438.5).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.48 (L-Ala),
L/D = 100/0; Rt = 10.88 (L-Leu), L/D = 100/0.
Synthesis of Fmoc-Ala-Ala-OMe (12l)
Starting materials: Fmoc-Ala-OH (0.329 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Fmoc-Ala-Ala-OMe (0.361 g, 91%),
mp = 189-190oC, lit. [49] mp = 195-196oC. Anal. RP-HPLC (50–100%B in 30 min): Rt = 8.35 min, purity
98%.
IR (film/NaCl): ν = 3119, 3008, 1740, 1718, 1675, 1504, 1450, 1376, 1314 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.40 (d, 3H + 3H, J = 7.5 Hz, CH3-CH-); 3.74 (s, 3H, CH3O-); 4.20 (t,
1H, J = 6.5 Hz, -CHCH2O); 4.20-4.30 (m, 1H, CH3CH); 4.40 (d, 2H, J = 6.5 Hz, -CHCH2O); 4.58 (q, 1H, J =
7.5 Hz, CH3-CH); 5.45 (broad d, 1H, J = 7 Hz, NHCOO); 6.54 (broad d, 1H, NH); 7.31 (t, 2H, J = 7 Hz,
arom.); 7.43 (t, 2H, J = 7 Hz, arom.); 7.60 (d, 2H, J = 7 Hz, arom.); 7.76 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.1, 17.5, 47.1, 47.8, 50.5, 52.2, 66.9, 119.9, 125.1, 127.1, 128.6,
142.2, 155.9, 172.3, 172.9 [ppm].
LC/MS: 397.5 ([M+H]+, C22H24N2O5 ; calc. 396.4).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.66 (L-Ala),
L/D = 100/0.
Synthesis of Fmoc-Ala-Phe-OMe (12m)
Starting materials: Fmoc-Ala-OH (0.329 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Phe-OMe*HCl (0.216 g, 1 mmol). Product: Fmoc-Ala-Phe-OMe (0.416 g, 88%),
mp = 159-160oC, lit. [49] mp = 160oC.
Anal. RP-HPLC (50–100%B in 15 min): Rt = 10.95 min, purity 98%.
IR (film/NaCl): ν = 3678, 3616, 3430, 3012, 2407, 1742, 1719, 1680, 1603, 1502, 1448, 1363 [cm-
1].
1H-NMR (250 MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.5 Hz, CH3-CH-); 3.08, 3.15 (d AB system, 2H, J1 =
14 Hz, J2 = 6 Hz, -CH2-C6H5); 3.71 (s, 3H, CH3O-); 4.20 (t, 1H, J = 7.5 Hz, -CHCH2O-); 4.15-4.40 (m, 1H,
CH); 4.42 (d, 2H, J = 7.5 Hz, -CHCH2O); 4.85 (q, 1H, J = 6 Hz, -CH2CH-); 5.30 (broad d, 1H, J = 7 Hz, -
NHCOO); 6.42 (broad d, 1H, J = 6.5 Hz, -NHCOO); 7.08-7.72 (m, 13H, arom.) [ppm].
This article is protected by copyright. All rights reserved.

13C-NMR (75 MHz, CDCl3): δ = 17.5, 37.6, 47.1, 50.5, 52.3, 53.2, 66.9, 119.9, 125.1, 127.1, 128.4,
128.7, 128.9, 129.1, 142.2, 155.9, 171.3, 172.2[ppm].
+
LC/MS: 473.6 ([M+H]+, C28H28N2O5 ; calc. 472.5).
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.28 (L-Ala),
L/D = 100/0; Rt = 22.16 (L-Phe), L/D = 100/0.
Synthesis of Fmoc-Pro-Ala-OMe (12n)
Starting materials: Fmoc-Pro-OH (0.337 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Fmoc-Pro-Ala-OMe (0.414 g, 98%),
mp = 90-92oC, lit. [50] mp = 92-94oC. Anal. RP-HPLC (50–100%B in 10 min): Rt = 8.90 min, purity 99%.
IR (film/NaCl): ν = 3350, 3170, 3100, 1740, 1670, 1530, 1430, 1360, 1340, 1270, 1210 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.37 (d, 3H, J = 6 Hz, CH3-CH-); 1.60-2.50 (m, 4H, -CH2-CH2-); 3.40-
3.80 (m, 2H, N-CH2-); 3.72 (s, 3H, CH3-O-); 4.15-4.45 (m, 4H, -O-CH2-CH- + -O-CH2-CH- + N-CH-); 4.53
(qu, 1H, J = 6 Hz, CH3CH); 7.08 (broad s, 1H, NH); 7.31 (t, 2H, J = 7 Hz, arom.); 7.40 (t, 2H, J = 7 Hz,
arom.); 7.57 (broad d, 2H, J = 7 Hz, arom.); 7.76 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 23.0, 28.6, 46.2, 47.1, 47.8, 52.2, 58.8, 67.5, 119.9, 125.1,
127.1, 128.6, 142.2, 154.5, 172.0, 172.9 [ppm].
+
LC/MS: 423.6 ([M+H]+, C24H26N2O5 ; calc. 422.5).
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 4.91 (L-Ala),
L/D = 100/0; Rt = 11.67 (L-Pro), L/D = 100/0.
Synthesis of Fmoc-Asp(OtBu)-Ala-OMe (12o)
Starting materials: Fmoc-Asp(tBu)-OH (0.412 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Fmoc-Asp(tBu)-Ala-OMe
(0.382 g, 77%), oil. Anal. RP-HPLC (50–100%B in 15 min): Rt = 11.26 min, purity 98%.
IR (film/NaCl): ν = 3410, 3090, 2990, 1750, 1740, 1710, 1675, 1540, 1450, 1375 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.40 (d, 3H, J = 7 Hz, CH3-CH-); 1.46 (s, 9H, -OC(CH3)3); 2.57 (dd,
1H, J1 = 17 Hz, J2 = 7 Hz, -OCO-CH2-CH-); 2.92 (dd, 1H, J1 = 17 Hz, J2 = 4 Hz, -OCO-CH2-CH-); 3.73 (s, 3H,
CH3O-); 4.26 (t, 1H, J = 7 Hz, -CH-CH2O-); 4.42 (d, 2H, J = 7 Hz, -CH-CH2O-); 4.48-4.60 (m, 2H, CH3CH- +
OCO-CH2-CH-); 5.97 (d, 1H, J = 7 Hz, NH); 7.07 (d, 1H, J = 5 Hz, NH); 7.31 (t, 2H, J = 7 Hz, arom.); 7.41
(t, 2H, J = 7 Hz, arom.); 7.58 (d, 2H, J = 7 Hz, arom.); 7.77 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 28.1, 37.9, 47.1, 47.8, 52.4, 53.6, 66.9, 81.4, 119.9, 125.1,
127.1, 128.6, 142.2, 155.5, 172.3, 172.5, 172.9 [ppm].
+
LC/MS: 497.7 ([M+H]+, C27H32N2O7 ; calc. 496.6).
This article is protected by copyright. All rights reserved.

GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.13 (L-Ala),
L/D = 100/0; Rt = 14.13 (L-Asp), L/D = 100/0.
Synthesis of Fmoc-Ser(tBu)-Ala-OMe (12p)
Starting materials: Fmoc-Ser(tBu)-OH (0.383 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Fmoc-Ser(tBu)-Ala-OMe
(0.412 g, 88%), mp = 83-84oC, lit. [50] mp = 84-86oC. Anal. RP-HPLC (50–100%B in 15 min): Rt = 11.38
min, purity 96%.
IR (film/NaCl): ν = 3410, 3320, 2990, 1740, 1720, 1670, 1480, 1440, 1375, 1210 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.24 (s, 9H, -OC(CH3)3); 1.45 (d, 3H, J = 7 Hz, CH3-CH-); 3.39 (dd,
1H, J1 = 9 Hz, J2 = 6 Hz, -CH-CH2-O-); 3.75 (s, 3H, CH3O-); 3.80 (dd, 1H, J1 = 9 Hz, J2 = 6 Hz, -CH-CH2-O-);
4.20-4.25 (m, 2H, -CHCH2O- +-NH-CH-CH2O); 4.39 (d, 2H, J = 7 Hz, -CHCH2O); 4.58 (qu, 1H, J = 7 Hz,
CH3CH-); 5.79 (broad d, 1H, J = 4 Hz, NH); 7.30 (t, 2H, J = 7 Hz, arom.); 7.39 (t, 2H, J = 7 Hz, arom.);
7.45 (broad m, 1H, NH); 7.60 (d, 2H, J = 7 Hz, arom.); 7.75 (d, 2H, J = 7 Hz, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 27.6, 38.3, 47.1, 47.8, 52.3, 66.9, 69.3, 73.5, 119.9, 125.1,
127.1, 128.6, 142.2, 155.5, 171.3, 172.9 [ppm].
LC/MS: 469.6 ([M+H]+, C26H32N2O6 ; calc. 468.6).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 4.69 (D-Ala), Rt
= 5.16 (L-Ala), L/D = 98.8/1.2; Rt = 10.32 (L-Ser), L/D = 100/0.
Synthesis of Fmoc-Thr(tBu)-Ala-OMe (12q)
Starting materials: Fmoc-Thr(tBu)-OH (0.396 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Fmoc-Thr(tBu)-Ala-OMe
(0.362 g, 75%), mp = 148-150oC, lit. [50] mp = 149-151oC. Anal. RP-HPLC (50–100%B in 15 min): Rt =
13.03 min, purity 95%.
IR (film/NaCl): ν = 3380, 3290, 1755, 1720, 1675, 1535, 1480, 1280, 1220 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.10 (d, 3H, J = 6 Hz, (CH3-CH-O-); 1.31 (s, 9H, -OC(CH3)3); 1.44 (d, 3H, J
= 7 Hz, CH3-CH-); 3.76 (s, 3H, CH3O-); 4.10-4.25 (m, 3H, -CHCH2O- + CH-O-CH-CH-NH); 4.38 (d, 2H, J =
7 Hz, -CHCH2O); 4.52 (qu, 1H, J = 7 Hz, CH3CH-); 6.00 (d, 1H, J = 4 Hz, NH); 7.24 (m, 1H, NH); 7.30 (t,
2H, J = 7 Hz, arom.); 7.39 (t, 2H, J = 7 Hz, arom.); 7.60 (d, 2H, J = 7 Hz, arom.); 7.75 (d, 2H, J = 7 Hz,
arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.2, 17.7, 28.3, 47.1, 47.8, 52.3, 66.9, 72.8, 73.7, 119.9, 125.1, 127.1,
128.6, 142.2, 155.5, 170.6, 172.9 [ppm].
+
LC/MS: 483.6 ([M+H]+, C27H34N2O6 ; calc. 482.6).
This article is protected by copyright. All rights reserved.

GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 4.67 (D-Ala), Rt
= 5.11 (L-Ala), L/D = 98.8/1.2; Rt = 7.34 (L-Thr), L/D = 100/0.
Synthesis of Boc-Aib-Ala-OMe (12r)
Starting materials: Boc-Aib-OH (0.203 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Boc-Aib-Ala-OMe (0.213 g, 74%), mp
= 194-96oC, lit. [51] mp = 95-95.5oC. Anal. RP-HPLC (50–100%B in 15 min): Rt = 10.22 min, purity
99%.
IR (film/NaCl): ν = 3320, 1756, 1688, 1668, 1656, 1516, 1368, 1280, 1252, 1168, 1088, 772
[cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.44 (s, 9H, (CH3)3-C-O-); 1.48 (s, 3H, CH3-CH-); 1.51 (s, 6H, CH3C-
CH3); 3.72 (s, 3H, -COO-CH3); 4.55-4.64 (m, 1H, -CH-COOCH3) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 25.1, 28.2, 48.1, 52.1, 79.6, 58.1, 156.8, 172.9, 175.6
[ppm].
LC/MS: 289.96 ([M+H]+, C13H25N2O5 ; calc. 288.35).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 6.49 (Aib); Rt =
5.11 (L-Ala), L/D = 100/0.
Synthesis of Boc-L-Trp-Ala-OMe (12s)
Starting materials: Boc-L-Trp-OH (0.304 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Boc-L-Trp-Ala-OMe (0.276 g, 71%),
mp = 110-112oC, lit. [52] mp = 112-113oC. Anal. RP-HPLC (50–100%B in 15 min): R t = 12.81 min,
purity 85%.
IR (film/NaCl): ν = 3344,1668, 1504, 1468, 1456, 1368, 1216, 1192, 1164, 1056, 792, 664 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.26 (d, 3H, J = 7 Hz, CH3-CH-); 1.43 (s, 9H, (CH3)3-C-O-); 3.12-3.38
(m, 2H, CH2-); 3.66 (s, 3H, -COO-CH3); 4.40-4.51 (m, 1H, -NHCH-CH3); 5.04-5.21 (m, 1H, CH-); 7.09-
7.37 + 7.64-7.67 (m, 5H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 27.7, 28.2, 47.8, 52.0, 53.6, 80.0, 109.8, 111.6, 118.7,
120.2, 122.2, 124.4, 127.6, 136.1, 155.3, 172.5, 172.9, [ppm].
LC/MS: 390.96 ([M+H]+, C20H28N3O5 ; calc. 389.46).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.11 (L-Ala),
L/D = 100/0.
This article is protected by copyright. All rights reserved.

Synthesis of Boc-L-Ala-L-Phe-OMe (12t)
Starting materials: Boc-L-Ala-OH (0.189 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA (0.264
mL, 1.5 mmol) and H-Phe-OMe*HCl (0.238 g, 1 mmol). Product: Boc-L-Ala-L-Phe-OMe (0.238 g, 68%),
mp = 79-83oC, lit. [53] mp = 80oC. Anal. RP-HPLC (50–100%B in 15 min): Rt = 10.97 min, purity 99%.
IR (film/NaCl): ν = 3312, 2976, 1680, 1516, 1444, 1368, 1312, 1164, 1048, 756, 704, 664
[cm-1].
1H-NMR (250 MHz, CDCl3): δ = 1.29 (d, 3H, J = 7 Hz, CH3-CH-); 1.43 (s, 9H, (CH3)3-C-O-); 3.03-3.23
(m, 2H, CH2-C6H5); 3.68 (s, 3H, -COO-CH3); 4.06-4.19 (m, 1H, -NH-CH-CH3); 4.84 (q, 1H, J = 6 Hz, CH-
CH2-C6H5); 6.85 (broad d, 1H, -NH-); 7.09-7.27 (m, 5H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 28.1, 38.4, 47.8, 52.3, 55.6, 80.0, 128.7, 128.9, 129.1,
155.3, 171.2, 172.9 [ppm].
+
LC/MS: 351.94 ([M+H]+, C18H27N2O5 ; calc. 350.42).
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 5.11 (L-Ala),
L/D = 100/0; Rt = 22.16 (L-Phe), Rt = 22.36 (D-Phe) L/D = 99/1.
Synthesis of Boc-L-Orn(Z)-L-Leu-OMe (12u)
Starting materials: Boc-L-Orn(Z)-OH (0.366 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Leu-OMe*HCl (0.182 g, 1 mmol). Product: Boc-L-Orn(Z)-L-Leu-OMe
(0.439 g, 89%), mp = 84-86oC, lit. [54] mp = 85-88oC. Anal. RP-HPLC (50–100%B in 15 min): Rt = 11.05
min, purity 100%.
IR (film/NaCl): ν = 3583, 2620, 3447, 3019, 2976, 2400, 1707, 1517, 1424 [cm-1].
1H-NMR (250 MHz, CDCl3): δ = 0.86 (d, 6H, J = 8.5 Hz, (CH3)2-CH-); 1.30-2.00 (m, 7H, -CH2CH- + -
CH2CH2CH2CH-); 1.43 (s, 9H, (CH3)3C-); 3.15 (q, 2H, J = 4,5 Hz, -NH-CH2CH2CH2CH-); 3.58 (m, 1H, -CH2-
CH-CONH-); 3.69 (s, 3H, -COO-CH3); 4.55 (q, 1H, J = 4.0 Hz, -CONH-CH-CH2-); 4.95 (broad s, 1H, NH);
5.07 (AB, 2H, J = 8.5 Hz, -CH2O-); 5.21 (m, 1H, NH); 6.95 (broad s 1H, NH); 7.35 (s, 5H, arom.) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 22.6, 24.8, 25.7, 28.2, 28.7, 40.0, 40.4, 52.1, 53.5, 53.8, 66.5, 80.0,
127.9, 128.5, 128.9, 155.3, 156.3, 172.5, 172.8 [ppm].
+
LC/MS: 494.92 ([M+H]+, C25H40N3O7 ; calc. 493.61).
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 11.03 (L-Orn)
L/D = 100/0; Rt = 10.88 (L-Leu), L/D = 100/0.
This article is protected by copyright. All rights reserved.

Synthesis of Boc-Ala-Gly-Phe-OMe (13a)
Starting materials: Boc-Ala-Gly-OH (0.246 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Phe-OMe*HCl (0.238 g, 1 mmol). Product: Boc-Ala-Gly-Phe-
OMe (0.318 g, 78%), oil, [α]D20 = -25 (c = 1.0, CHCl3). Anal. RP-HPLC (50–100%B in 15 min): Rt =
8.82 min, purity 90%.
IR (film/NaCl): ν = 3136, 3064, 3048, 2976, 1680, 1632, 1360, 1160, 1048, 856, 760, 704,
648 [cm-1].
1H-NMR (CDCl3): δ = 1.28 (d, 3H, J = 8 Hz, CH3 Ala); 1.43 (s, 9H, (CH3)3C Boc); 3.19 (t, 2H, J = 6 Hz,
-CH2-CH- Phe); 3.90 (s, 3H, -COO-CH3); 3.93-3.97 (m, 2H, -CH2- Gly); 4.36 (q, 1H, J = 5 Hz, -CH- Ala);
4.82 (q, 1H, J = 6 Hz, -CH- Phe), 5.13 (broad d, 1H, -NH-); 6.97 (broad d, 1H, -NH-); 7.08-7.31 (m, 5H,
C6H5- Phe) [ppm].
LC/MS: 408.97 ([M+H]+, C20H30N3O6 ; calc. 407.47).
+
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 4.55 (L-Ala),
L/D = 100/0; Rt = 6.78 (Gly); Rt = 20.11 (D-Phe), Rt = 20.38 (L-Phe), L/D = 98/2.
Synthesis of Boc-Ala-Gly-Ala-OMe (13b)
Starting materials: Boc-Ala-Gly-OH (0.246 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Boc-Ala-Gly-Ala-
20
20
OMe (0.268 g, 81%), oil, [α]D = -15 (c = 1.0, MeOH), lit. [55] α]D = -7 (c = 1.0, CH2Cl2). Anal. RP-
HPLC (50–100%B in 15 min): Rt = 9.48 min, purity 91%.
IR (NaCl/film): ν = 3352, 3128, 2984, 1748, 1632, 1512, 1360, 1164, 1028, 856, 736, 648
[cm-1].
1H-NMR (CDCl3): δ = 1.27 (d, 3H, J = 8 Hz, CH3 Ala); 1.36 (d, 3H, J = 8 Hz, CH3 Ala); 1.41 (s, 9H,
(CH3)3C- Boc);3.71-3.89 (m, 2H, -CH2- Gly); 3.94 (s, 3H, -COO-CH3);4.38-4.41 (m, 1H, -CH- Ala); 4.59-
4.85 m, 1H, -CH- Ala); 6.01 (broad d, 1H, -NH-) [ppm].
+
LC/MS: 332.89 ([M+H]+, C14H26N3O6 ; calc. 331.37).
GC: the hydrolysate was derivatized according to the standard procedure; chromatography
conditions: temperature 60-150oC, 4oC/min, 150oC, 5 min, 150-180oC, 40oC/min; Rt = 4.66 (L-Ala),
L/D = 100/0; Rt = 6.72 (Gly).
Synthesis of Boc-Ala-Trp-Ala-OMe (13c)
Starting materials: Boc-Ala-Trp-OH (0.375 g, 1 mmol); DMT/NMM/TsO- (0.413 g, 1 mmol); DIPEA
(0.264 mL, 1.5 mmol) and H-Ala-OMe*HCl (0.139 g, 1 mmol). Product: Boc-Ala-Trp-Ala-
This article is protected by copyright. All rights reserved.