Enantioselective solid-phase peptide synthesis using traceless chiral coupling reagents and racemic amino acids

Article abstract of DOI:10.1002/hlca.201200426

The enantioselective condensing reagent 4,6-dimethoxy-1,3,5-triazine (DMT)/strychnine/BF-₄ was obtained by treatment of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) with strychnine tetrafluoroborate. The reagent was useful under typical conditions of solid-phase peptide synthesis (SPPS) with enantiomerically homogeneous substrates. By SPPS, desired dipeptides were obtained in 84-94% yield using 4 equiv. of racemic Fmoc-Ala, Fmoc-Phe, and/or Fmoc-Tyr for 1 equiv. of Wang resin loaded with Gly, Ala, Leu, Phe, Glu(^tBu), and/or Pro, respectively. For all three Fmoc-protected amino acids, the configuration of the enantiomer preferred under SPPS conditions was independent of the structure of the acylated component and identical to that established in condensations proceeding in solution. In all cases, the enantiomer ratios L/D (er) were in a similar range, and varied from 9: 92 to 2: 98 for alanine, and from 90: 10 to 100: 0 for aromatic amino acids. The synthesis of Ac-L-Lys(Ac)-D-Ala-D-Ala-OH from racemic Fmoc-Ala gave an L/D ratio of 10: 90 for the esterification of Wang resin, and 0: 100 for the formation of peptide bonds.

Full text of DOI:10.1002/hlca.201200426

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Helvetica Chimica Acta – Vol. 95 (2012)
Enantioselective Solid-Phase Peptide Synthesis Using Traceless Chiral
Coupling Reagents and Racemic Amino Acids
by Beata Kolesinska*, Katarzyna Kasperowicz-Frankowska, Justyna Fraczyk,
and Zbigniew J. Kaminski*
Institute of Organic Chemistry, Technical University of Łodz, Zeromskiego 116, PL-90-924 Łodz
(phone: þ 48-42-6313151; fax: þ 48-42-6365530; e-mail: zbigniew.kaminski@p.lodz.pl,
beata.kolesinska@p.lodz.pl)
Dedicated to Prof. Dieter Seebach on the occasion of his 75th birthday
The enantioselective condensing reagent 4,6-dimethoxy-1,3,5-triazine (DMT)/strychnine/BFꢀ4 was
obtained by treatment of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) with strychnine tetrafluorobo-
rate. The reagent was useful under typical conditions of solid-phase peptide synthesis (SPPS) with
enantiomerically homogeneous substrates. By SPPS, desired dipeptides were obtained in 84 – 94% yield
using 4 equiv. of racemic Fmoc-Ala, Fmoc-Phe, and/or Fmoc-Tyr for 1 equiv. of Wang resin loaded with
Gly, Ala, Leu, Phe, Glu(tBu), and/or Pro, respectively. For all three Fmoc-protected amino acids, the
configuration of the enantiomer preferred under SPPS conditions was independent of the structure of the
acylated component and identical to that established in condensations proceeding in solution. In all cases,
the enantiomer ratios l/d (er) were in a similar range, and varied from 9 :92 to 2 :98 for alanine, and from
90 :10 to 100 :0 for aromatic amino acids. The synthesis of Ac-l-Lys(Ac)-d-Ala-d-Ala-OH from racemic
Fmoc-Ala gave an l/d ratio of 10 :90 for the esterification of Wang resin, and 0 :100 for the formation of
peptide bonds.
1. Introduction. – Methods of Enantioselective Acylation. Ready access to a whole
range of chiral building blocks is essential to derive full profits from the systematic
exploration of a diversity of peptide structures by using the latest methods of
combinatorial chemistry and high throughput screening. The classical approach based
on asymmetric synthesis or resolution of a racemic precursor seems to be non-optimal,
because most substrates are used in minute amounts in single experiments only, and
usually they are discarded after a preliminary selection procedure as not promising.
Therefore, an approach based on the enantiomer-differentiating transformation of
racemic substrates, which are usually easily available, would be welcome as more
advantageous and less time-consuming than the long-established procedures based on
racemate resolution or asymmetric synthesis. Such an approach would be considered
the most convenient procedure based on coupling chiral building blocks, such as amino
acids used for the construction of complex peptide molecules. Although several chiral
coupling reagents and chiral acylation catalysts have been proposed for the synthesis of
enantiomerically enriched peptides directly from racemic substrates, including
optically active N-hydroxysuccinimide [1 – 6], diacylamines [7 – 14], chiral 4-(dime-
thylamino)pyridine (DMAP) analogs [15 – 23], 1-methyl-1H-imidazole [24 – 30],
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 95 (2012)
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benzotetramisol [31], tertiary amines [32 – 36] and phosphines [37 – 45], and others
[46 – 55] as chiral auxiliaries, so far none of the proposed enantioselective coupling
methods has been acceptable for peptide synthesis because of the unpredictable
configuration, enantiomeric purity, and yield of the final product.
The unpredictability of the enantioselective synthesis of peptides is caused by the
fairly complex mechanism of this process, consisting of two subsequent stages. In the
first one, the carboxylic group is activated, followed by aminolysis of the activated
species in the second step. In both of these elementary stages, tetrahedral intermediates
are formed with additional stereogenic centers at the C-atom of the former carboxylic
group. Moreover, both the formation and decay of the tetrahedral intermediates,
proceeding via multiple transition states, depend on the multifaceted matching/
dismatching effects of all the stereogenic centers of all the reacting molecules.
2. Chiral 1,3,5-Triazine-Based Coupling Reagents for Enantiomerically Enriched
(Pure) Peptides Directly from Racemic Substrates. – Recently, we solved the problem
of unpredictable enantioselective synthesis of peptides from racemic substrates. This
was accomplished by substantial simplification of interactions between stereogenic
centers. To achieve this, traceless enantioselective coupling reagents were designed
with the chiral fragment departing after the activation of a carboxylic component.
According to this concept, such reagents are prepared by temporary attachment of the
chiral fragment to a classical peptide-bond-forming agent. Thus, after selection of the
enantiomer at the activation stage and departure of the chiral component, the activated
intermediate is converted to a well-known form of a conventional acylating species. To
date, the only traceless reagent according to the concept presented above has been
obtained by modification of the 1,3,5-triazine-based coupling reagent 2 with the chiral
fragment integrated in the ammonium leaving group L*, departing as HL* after
activation with the formation of the ꢂclassicꢃ 1,3,5-triazin-2-yl ꢂsuperactiveꢃ ester 3 [56 –
59] (Scheme 1).
Scheme 1. Enantioselective Activation of the Carboxylic Component with the Traceless Coupling Reagent
4 Affords the Activated Carboxylic Component 3 Identical to the Product of Activation with the Classic
Achiral Reagent 2

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Helvetica Chimica Acta – Vol. 95 (2012)
Thus, for every racemic carboxylic component, the results of synthesis may be
predicted from a single model experiment. On the other hand, any modification of the
chiral fragment necessary to access the preferred configuration or to improve
enantiomeric enrichment gives exactly the same acylating intermediate, which
significantly facilitates the synthetic protocol. Triazine coupling reagents containing
strychnine (DMT/strychnine/BFꢀ4 ) [60] or brucine (DMT/brucine/BFꢀ4 ) [61] as the
chiral fragment have proved highly effective in the synthesis of enantiomerically
enriched or even enantiomerically homogeneous peptides from racemic substrates in
the solution phase.
3. Attempts to Exploit of Chiral Traceless Coupling Reagents in the Solid-Phase
Peptide Synthesis (SSPS). – It is well-known that enantiomeric enrichment obtained in
an enantioselective process is substantially enhanced by a greater excess of the racemic
substrate. However, in syntheses proceeding in solution, the excess of the substrate
used may severely impede the isolation of the final product. Therefore, one can
presume that this drawback could be completely eliminated under conditions of SPPS.
The application of a racemic substrate in a reasonably increased excess should enhance
enantioselectivity, unify enantiomeric enrichment for homologous substrates, and be
advantageous for the final yield as well purity of the product. To verify the potential of
this approach, the enantioselective coupling reagent 6 was obtained by treatment of 2-
chloro-4,6-dimethoxy-1,3,5-triazine (CDMT; 1) with strychnine tetrafluoroborate as
described in [60] and used in SPPS. In preliminary experiments, we confirmed the
versatility of 6 under conditions of the SPPS standard protocol involving optically pure
N-protected amino acids, an augmented amount of tertiary amine, and a variety of
additives [62] (Scheme 2).
Scheme 2. The Selection of Conditions for Peptide-Bond Formation Using Chiral Triazine Coupling
Reagents in SPPS

Helvetica Chimica Acta – Vol. 95 (2012)
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In the study, enantiomerically pure amino acids prone to racemization, i.e., Fmoc-l-
Phe-OH (7b), Fmoc-l-Ser(tBu)-OH (7c), and Fmoc-l-His(Trt)-OH (7d), were used as
substrates1) (Table 1).
Table 1. Solid-Phase Peptide Synthesis Using DMT/Strychnine/BFꢀ4 (6). In all reactions, 4 equiv. of N-
protected l-amino acids, 2 equiv. of DMT/strychnine/BFꢀ4 , 4 equiv. of tertiary amine, and 1 equiv. of
amino component (Val-Wang resin) were used.
Entry C-Comp. 7 Amine
Product 10
Yield er1) C-Comp. er1) N-Comp.
[%]
H-l-Ala-l-Val-OH (10aa) 91
Strychnine H-l-Ala-l-Val-OH (10aa) 90
EtNiPr2
H-l-Phe-l-Val-OH (10ba) 93
Strychnine H-l-Phe-l-Val-OH (10ba) 92
(l/d)
(l/d)
1
2
3
4
5
6
7
8
Ala (a)
Ala (a)
Phe (b)
Phe (b)
Ser (c)
Ser (c)
His (d)
His (d)
EtNiPr2
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
99.8 : 0.2
100 : 0
99.7: 0.3
99.8 : 0.2
99.5 : 0.5
99.6 : 0.4
EtNiPr2
Strychnine H-l-Ser-l-Val-OH (10ca)
EtNiPr2
H-l-Ser-l-Val-OH (10ca)
90
91
H-l-His-l-Val-OH (10da) 89
Strychnine H-l-His-l-Val-OH (10da) 88
It has been found that the presence of strychnine or DIPEA (EtNiPr2) does not
alter the solid-phase coupling procedure with 6 as a reagent for peptide-bond
formation. In some cases, minimal racemization of C-components was observed;
however, rather than being the result of epimerization, this may be due to rather harsh
conditions of hydrolysis of peptides to amino acids, which was indispensable for the
evaluation of their enantiomeric composition by gas chromatography on chiral
stationary phase.
The absence of racemization of the acylating components in the presence of DMT/
strychnine/BFꢀ4 (6) under SPPS conditions prompted us to apply 6 as a chiral coupling
reagent in the synthesis of peptides directly from racemic N-protected amino acids
under such conditions. The usefulness of 6 was then evaluated by the SPPS of
dipeptides using racemic Fmoc-alanine, Fmoc-phenylalanine, and Fmoc-tyrosine as
starting materials. In all cases syntheses were performed on Wang resin2). The process
involved two steps: i) activation of the racemic Fmoc-protected C-component
(2 equiv.) by using DMT/strychnine/BFꢀ4 (6) to yield enantiomerically enriched active
ester 3; ii) acylation of an N-component anchored to Wang resin 8 (Scheme 3).
The configurational preferences and enantiomeric enrichment of the selected
racemic N-protected substrates in syntheses proceeding in solution were determined
previously in independent condensation experiments. As expected, for all three amino
acids the enantiomers kinetically selected under SPPS conditions were independent of
the structure of the acylated component and were the same as those established
previously in condensations proceeding in solution [60], which opens a new venue for
the synthetic application of traceless chiral coupling reagents (Table 2).
1
)
The configuration and optical purity of isolated products were determined after hydrolytic
degradation to amino acids, and the subsequent derivatization and separation of enantiomers by
GC on a Chirasil Val capillary column.
2
)
Preliminary results were published in [63].

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Helvetica Chimica Acta – Vol. 95 (2012)
Scheme 3. Synthesis of Dipeptides from Racemic Fmoc-Protected Amino Acids Using DMT/Strychnine/
BFꢀ4 (6) under SPPS Conditions
Table 2. Synthesis of Enantiomerically Enriched Peptides Using DMT/Strychnine/BFꢀ4 (6) Directly from
Racemic Starting Materials. In all experiments, 4 equiv. of racemic N-protected amino acid (7), 2 equiv. of
DMT/strychnine/BFꢀ4 (6), 0.4 equiv. of strychnine, and 1 equiv. of amino acid anchored to Wang resin, 8,
were used; activation time of racemic C-component, 15 min.
Entry Racemic
C-Component 7
NH2-AaaꢀWang resin 8 Product prepared 10
Yield er1) N-Termini
[%] amino acid
(l/d)
1
2
3
4
5
Fmoc-Ala-OH (7a) NH2-Glyꢀ (8b)
H-d-Ala-Gly-OH (10ab) 93
H-d-Ala-l-Leu-OH (10ac) 91
H-d-Ala-l-Phe-OH (10ad) 92
2 : 98
5 : 95
8 : 92
3 : 97
4 : 96
NH2-l-Leuꢀ (8c)
NH2-l-Pheꢀ (8d)
NH2-l-Glu(OtBu)ꢀ (8e) H-d-Ala-l-Glu-OH (10ae) 89
NH-l-Proꢀ (8f)
H-d-Ala-l-Pro-OH (10af) 94
6
7
8
9
10
Fmoc-Phe-OH (7b) NH2-Glyꢀ (8b)
H-l-Phe-Gly-OH (10bb) 90
H-l-Phe-l-Leu-OH (10bc) 92
H-l-Phe-l-Ala-OH (10bg) 94
90 : 10
100 : 0
98 : 2
98 : 2
96 : 4
NH2-l-Leuꢀ (8c)
NH2-l-Alaꢀ (8g)
NH2-l-Glu(OtBu)ꢀ (8e) H-l-Phe-l-Glu-OH (10be) 88
NH-l-Proꢀ (8f)
H-l-Phe-l-Pro-OH (10bf) 90
11
12
13
14
15
16
Fmoc-Tyr-OH (7e) NH2-Glyꢀ (8b)
H-l-Tyr-Gly-OH (10eb)
89
89 : 11
100 : 0
100 : 0
98 : 2
100 : 0
99 : 1
NH2-l-Leuꢀ (8c)
H-l-Tyr-l-Leu-OH (10ec) 93
H-l-Tyr-l-Ala-OH (10eg) 92
H-l-Tyr-l-Phe-OH (10ed) 91
NH2-l-Alaꢀ (8g)
NH2-l-Pheꢀ (8d)
NH2-l-Glu(OtBu)ꢀ (8e) H-l-Tyr-l-Glu-OH (10ee) 88
NH-l-Proꢀ (8f)
H-l-Tyr-l-Pro-OH (10ef) 89
The versatility of the traceless chiral coupling reagents was further confirmed in the
synthesis of the peptide Ac-l-Lys(Ac)-d-Ala-d-Ala-OH (11)3), which is a component
3
)
The cell wall of many bacteria consist of peptides containing d-Ala-d-Ala fragment, which is the
target for glycopeptide antibiotics, often regarded as drugs of last resort in Gram-positive bacteria
infections [65 – 67].

Helvetica Chimica Acta – Vol. 95 (2012)
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of bacterial cell walls. The synthesis of 11 was achieved on Wang resin using racemic
Fmoc-Ala-OH and enantiomerically homogeneous Fmoc-l-Lys(Fmoc)-OH
(Scheme 4).
Scheme 4. Synthesis of Ac-l-Lys(Ac)-d-Ala-d-Ala-OH Using DMT/Strychnine/BFꢀ4 (6)
The first stage of synthesis involved the anchoring of alanine residues to Wang resin.
It is already known that anchoring N-protected amino acids to HO resins via ester
bonds proceeds less readily than via the formation of amide bonds, and requires a
stoichiometric amount of tertiary amine in order to activate the OH group of the solid
support [64]. In several cases, a basic environment is considered an important feature
promoting the partial racemization of the activated component. As expected, the
attachment of the first alanine residue to the resin proceeded enantioselectively with
the d-Ala/l-Ala ratio of 90 :10 (Table 3).
Table 3. Synthesis of Ac-l-Lys(Ac)-d-Ala-d-Ala-OH (11) Using DMT/Strychnine/BFꢀ4 (6) Directly from
Racemic Fmoc-Ala-OH
Entry
Step
Product
er1) Ala (d/l)
1
2
3
I
II
III
Fmoc-Ala-Wang resin
Fmoc-Ala-Ala-Wang resin
Fmoc-l-Lys(Fmoc)-Ala-Ala-Wang resin
90 : 10
95 : 5
95 : 5
The second step of the synthesis was also based on the enantiomer-differentiating
activation of racemic Fmoc-Ala-OH, yielding the dipeptide Fmoc-Ala-Ala anchored to
the Wang resin. In this step, the enantiomer ratio (er) determined as the total for both
Ala residues was d/l 95 :5. The obtained result shows that the incorporation of the
second alanine residue in the peptide chain occurred in an almost completely

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Helvetica Chimica Acta – Vol. 95 (2012)
enantiospecific way, yielding the product of the expected dd configuration. The last
synthetic step was completed in the presence of the classical achiral triazine-based
coupling reagent DMT/NMM/BFꢀ4 (2) [59] using optically homogenous Fmoc-l-
Lys(Fmoc)-OH. An examination of the enantiomeric composition of the final product
isolated after acetylation and cleavage from the resin reconfirmed the expected
configurations for all stereogenic centers.
4. Conclusions and Outlook. – The application of chiral (1,3,5-triazin-2-yl)ammo-
nium tetrafluoroborates has paved the way for using racemic N-protected amino acids
as substrates in SPPS. The most advantageous aspect of this approach is the
predictability of SPPS coupling conditions, configuration, and the optical purity of
the final product based on a simple coupling experiment, even if the model experiment
is performed in solution. Moreover, SPPS involving excess acylating reagent should be
regarded as particularly favorable for stereoisomer-differentiating reactions due to the
inherent advantage of this methodology under excess conditions.
This study was supported by the Polish State Committee for Scientific Research under Project PBZ-
KBN-126/T09/15.
Experimental Part
General. Abbreviations. DMAP, 4-(dimethylamino)pyridine; Fmoc, [(9H-fluoren-9-yl)methoxy]-
carbonyl; DMT/NMM/BFꢀ4 , 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluorobo-
rate; DMT/strychnine/BFꢀ4 , N-(4,6-dimethoxy-1,3,5-triazin-2-yl)strychninium tetrafluoroborate; HOBt,
1-hydroxybenzotriazole TFA, CF3COOH; TIPS, (i-Pr)3SiH; h.v., 0.1 – 0.01 Torr; TNBS, 2,4,6-trinitro-
benzenesulfonic acid.
GC: FID (H2/air); split 1:50; Chirasil l-Val cap. column (25 m ꢁ 0.25 mm); carrier gas, He; pressure,
135 kPa. 1H-NMR Spectra: Bruker Avance DPX 250 (250 MHz) spectrometer; d in ppm rel. to Me4Si as
internal standard, J in Hz. LC/MS Spectra: Dionex UltiMate 3000.
1. Peptide Synthesis. Amino acids were purchased from Novabiochem or Bachem. Wang resin and
Fmoc-Aaa-Wang resin were purchased from Novabiochem.
Manual Solid-Phase Peptide Synthesis (SPPS). All peptides were synthesized on Merck-Heidolph
Paraller Synthesis-1 by Fmoc methodology [68].
Coupling Procedure by Using DMT/Strychnine/BFꢀ4 (6; GP 1). Two equiv. of amino acid, 4 equiv. of
EtNiPr2 or strychnine, 2 equiv. of HOBt, and 2 equiv. of DMT/strychnine/BFꢀ4 (6) as a coupling reagent
were dissolved in DMF (3 – 5 ml), added to the resin (1 equiv.), and shaken for 30 min. The completion
of the reaction was monitored by TNBS [69] or Kaiser test [70].
Deprotection (GP 2). The Fmoc group was removed by a soln. of 20% piperidine in DMF (2 ꢁ
5 min).
Cleavage from the Resin (GP 3). The peptides were cleaved from the resin using TFA/Et3SiH/H2O
9.5 :2.5 :2.5 (ca. 2 ml/0.1 g of resin). Cleavage was performed during 2 h, then the resin was filtered off,
and the filtrate was evaporated. To the residue, Et2O was added to precipitate the peptide. The resulting
solid was filtered off, and washed with Et2O. The product was dissolved in H2O and lyophilized. The final
products were obtained as a TFA salts.
2. Enantiomer-Differentiating Solid-Phase Peptide Synthesis (SPPS) by Using DMT/Strychnine/BF4ꢀ
(6; GP 4). To the vigorously stirred soln. of DMT/strychnine/BFꢀ4 (6; 2 equiv.) in MeCN (3 ml), cooled
to 08, strychnine (0.40 equiv.) was added, and, after 5 min, the soln. was treated with the appropriate
racemic carboxylic acid (4 equiv.). The stirring was continued for additional 15 min, and then the soln.
was added to freshly prepared amino component (1 equiv.; Fmoc deprotection according GP 2)

Helvetica Chimica Acta – Vol. 95 (2012)
2091
anchored to the Wang resin. Shaking was continued for 1 h. After filtration of soln., the resin was washed
with DMF (3 ꢁ 5 ml) and CH2Cl2 (3 ꢁ 5 ml).
Anal. RP-HPLC. Performed on a Waters 600S HPLC system (Waters 2489 UV/VIS detector, Waters
616 pump type, Waters 717 plus Autosampler type; HPLC manager, Chromax) using a Vydac C18 column
(25 cm ꢁ 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 the UV detection at 220 nm; tR in min.
3. Hydrolysis of Products of Coupling to Amino Acid (GP 5). Peptide (5 mg) was treated with
redistilled, constant boiling HCl in the sealed tube at 1008 for 20 h. The soln. was concentrated to dryness,
and the residue was dissolved with redistilled H2O and concentrated again. Remaining salt was dried
overnight in a vacuum desiccator under P2O5 and then treated with anh. MeOH sat. with dry HCl (2 ml)
for 12 h at r.t. MeOH was evaporated, and the residue was dried overnight in a vacuum desiccator under
P2O5, then suspended in CH2Cl2 and treated with (CF3CO)2O (50 ml) for 12 h at r.t. The soln. was
analyzed on a Chirasil l-Val cap. column (25 m ꢁ 0.25 mm); split, 1:50; He was used as carrier gas.
H-l-Ala-l-Val-OH (10aa; Table 1, Entry 1). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-Ala-OH (47 mg, 0.15 mmol) in the
presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and EtNiPr2 (54 ml,
0.30 mmol; GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and
lyophilized to give 21 mg of 10aa as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 8.11 min, purity
> 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions: temp. 908,
4 min; 90 – 2008, 48/min; 2008, 3 min; tR 4.00 (l-Ala), 3.81 (d-Ala); l/d 100 :0; 5.19 (l-Val), 5.15 (d-Val);
l/d 100 :0. 1H-NMR (250 MHz, D2O): 0.95, 0.97 (dd, J ¼ 6.0, Me2CH); 1.44 (d, J ¼ 7.1, MeCH); 2.08 – 2.10
(m, Me2CH); 4.01 – 4.04 (m, 1 H, CHꢀCH); 4.59 (q, J ¼ 7.5, MeCH); 5.40 (d, J ¼ 8.5, NHCOO); 6.31 (d,
J ¼ 6, NH). LC/MS: 189.82 ([M þ H]þ, C8H17N2O3þ ; calc. 189.23).
H-l-Ala-l-Val-OH (10aa; Table 1, Entry 2). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-Ala-OH (47 mg, 0.15 mmol) in the
presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and strychnine
(100 mg, 0.30 mmol; GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and
lyophilized to give 20 mg of 10aa as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 8.12 min, purity
> 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions: temp. 908,
4 min; 90 – 2008, 48/min; 2008, 3 min; tR 4.00 (l-Ala), 3.81 (d-Ala); l/d 100 :0; 5.19 (l-Val), 5.15 (d-Val);
1
l/d 100 :0. H-NMR (250 MHz, D2O): identical to those described above. LC/MS: 189.82 ([M þ H]þ,
C8H17N2Oþ3 ; calc.189.23).
H-l-Phe-l-Val-OH (10ba; Table 1, Entry 3). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-Phe-OH (58 mg, 0.15 mmol) in the
presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and EtNiPr2 (54 ml,
0.30 mmol; GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and
lyophilized to give 26 mg of 10ba as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 9.11 min,
purity > 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions:
temp. 908, 4 min; 90 – 2008, 48/min; 2008, 3 min; tR 18.89 (l-Phe), 18.44 (d-Phe); l/d 99.8 :0.2; 5.19 (l-
Val), 5.15 (d-Val); l/d 100 :0. 1H-NMR (250 MHz, D2O): 0.94, 0.96 (dd, J ¼ 6.0, Me2CH); 2.12 (m, 1 H,
Me2CH); 3.10, 3.14 (d, AB system, J ¼ 14.0, 5.0, CHCH2Ph); 4.00 – 4.08 (m, 1 H, CHꢀCH); 4.85 (q, J ¼
6.5, 1 H, PhCH2ꢀCH); 5.75 (br., NH); 7.08 – 7.32 (m, 6 arom. H, NH). LC/MS: 265.47 ([M þ H]þ,
C14H21N2Oþ3 ; calc. 265.33).
H-l-Phe-l-Val-OH (10ba; Table 1, Entry 4). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-Phe-OH (58 mg, 0.15 mmol) in the
presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and strychnine
(100 mg, 0.30 mmol; GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and
lyophilized to give 26 mg of 10ba as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 9.12 min,
purity > 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions:

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temp. 908, 4 min; 90 – 2008, 48/min; 2008, 3 min; tR 18.88 (l-Phe), 18.43 (d-Phe); l/d ¼ 100 :0; 5.18 (l-Val),
5.15 (d-Val); l/d 100 :0. 1H-NMR (250 MHz, D2O): product identical to those described above. LC/MS:
265.47 ([M þ H]þ, C14H21N2Oþ3 ; calc. 265.33).
H-l-Ser-l-Val-OH (10ca; Table 1, Entry 5). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-Ser(tBu)-OH (57 mg, 0.15 mmol) in
the presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and EtNiPr2
(54 ml, 0.30 mmol; GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and
lyophilized to give 21 mg of 10ca as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 7.76 min, purity
> 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions: temp. 908,
4 min; 90 – 2008, 48/min; 2008, 3 min; tR 7.75 (l-Ser), 7.79 min (d-Ser); l/d 99.7:0.3; 5.18 (l-Val), 5.15 (d-
Val) ; l/d 100 :0. 1H-NMR (250 MHz, D2O): 0.94, 0.97 (dd, J ¼ 6.1, Me2CH); 2.12 (m, Me2CH); 3.36 (dd,
J ¼ 8.1, 4.0, 1 H, CHꢀCH2O); 3.81 (dd, J ¼ 8.1, 4.0, 1 H, CHꢀCH2O); 4.00 – 4.08 (m, 1 H, CHꢀCH);
4.19 – 4.25 (m, 1 H, NHꢀCHCH2O); 5.75 (br., NH). LC/MS: 204.21 ([M þ H]þ, C8H17N2O4þ ; calc.
204.23).
H-l-Ser-l-Val-OH (10ca; Table 1, Entry 6). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-Ser(tBu)-OH (57 mg, 0.15 mmol) in
the presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and strychnine
(100 mgl, 0.30 mmol; GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3)
and lyophilized to give 22 mg of 10ca as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 7.77 min,
purity > 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions:
temp. 908, 4 min; 90 – 2008, 48/min; 2008, 3 min; tR 7.75 (l-Ser), 7.78 (d-Ser); l/d ¼ 99.8 :0.2; 5.17 (l-Val),
1
5.14 (d-Val); l/d 100 :0. H-NMR (250 MHz, D2O): identical to those described above LC/MS: 204.22
([M þ H]þ, C8H17N2Oþ4 ; calc. 204.23).
H-l-His-l-Val-OH (10da; Table 1, Entry 7). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-His(Trt)-OH (93 mg, 0.15 mmol) in
the presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and EtNiPr2
(54 ml, 0.30 mmol) (GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and
lyophilized to give 25 mg of 10da as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 6.11 min,
purity > 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions:
temp. 908, 4 min; 90 – 2008, 48/min; 2008, 3 min; tR 30.36 (l-His), 30.41 (d-His); l/d 99.5 :0.5; 5.17 (l-Val),
5.14 (d-Val); l/d 100 :0. 1H-NMR (250 MHz, D2O): 0.94, 0.97 (dd, J ¼ 6.1, Me2CH); 2.12 – 2.15 (m,
Me2CH); 2.84, 3.03 (d, AB system, J ¼ 14.0, 4.0, 2 H, CHꢀCH2ꢀC¼CHꢀNH); 4.00 – 4.06 (m, 1 H,
CHꢀCH); 4.61 (q, J ¼ 6.5, 1 H, CHꢀCH2C3H3N2); 5.88 (br., NH); 7.78 (br., NH). LC/MS: 255.37 ([M þ
H]þ, C11H19N4Oþ3 ; calc. 255.29).
H-l-His-l-Val-OH (10da; Table 1, Entry 8). Fmoc-Val-Wang resin (100 mg, 0.75 mmol/g,
0.075 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-His(Trt)-OH (93 mg, 0.15 mmol) in
the presence of DMT/strychnine/BFꢀ4 (84 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol), and strychnine
(100 mg, 0.30 mmol; GP 1). After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and
lyophilized to give 26 mg of 10da as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min) tR 6.10 min, purity
> 98%. GC: hydrolysate has been derivatized according to GP 5; chromatography conditions: temp. 908,
4 min; 90 – 2008, 48/min; 2008, 3 min; tR 30.36 (l-His), 30.40 (d-His); l/d 99.6 :0.4; 5.16 (l-Val), 5.14 (d-
1
Val) ; l/d 100 :0. H-NMR (250 MHz, D2O): identical to those described above. LC/MS: 255.38 ([M þ
H]þ, C11H19N4Oþ3 ; calc. 255.29).
H-d-Ala-Gly-OH (10ab; Table 2, Entry 1). Fmoc-Gly-Wang resin (100 mg, 0.77 mmol/g,
0.077 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Ala-OH (96 mg, 0.308 mmol) in
the presence of DMT/strychnine/BFꢀ4 (86 mg, 0.154 mmol) and strychnine (9 mg, 0.028 mmol; GP 4).
After deprotection (GP 2) dipeptide was cleaved from the resin (GP 3) and lyophilized to give 19 mg of
10ab as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 7.26 min, purity > 98%. GC: hydrolyzate

Helvetica Chimica Acta – Vol. 95 (2012)
2093
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 5.19 (Gly), 4.13 (l-Ala), 3.79 (d-Ala); l/d 2 :98. 1H-NMR (250 MHz, D2O): 1.41 (d, J ¼
6.5, MeCH); 3.96 (d, J ¼ 4.0, NHꢀCH2COO); 4.39 (m, MeCH); 6.61 (br. s, NH). LC/MS: 147.55 ([M þ
H]þ, C5H11N2Oþ3 ; calc. 147.15).
H-d-Ala-l-Leu-OH (10ac; Table 2, Entry 2). Fmoc-l-Leu-Wang resin (100 mg, 0.86 mmol/g,
0.086 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Ala-OH (108 mg, 0.344 mmol) in
the presence of DMT/strychnine/BFꢀ4 (96 mg, 0.172 mmol) and strychnine (12 mg, 0.034 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 25 mg of
10ac as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 7.92 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 8.56 (l-Leu), 4.16 (l-Ala), 3.79 (d-Ala); l/d 5 :95. 1H-NMR (250 MHz, D2O): 0.91 (d, J ¼
6.0, Me2CH); 1.34 (d, J ¼ 7.1, MeCH); 1.45 – 1.7 (m, MeCH); 4.46 (qu, J ¼ 7.1, MeCH); 4.59 (dt, J ¼ 8.0,
4.5, CH2CH); 6.61 (d, J ¼ 5, NH). LC/MS: 203.38 ([M þ H]þ, C9H19N2O3þ ; calc. 203.26).
H-d-Ala-l-Phe-OH (10ad; Table 2, Entry 3). Fmoc-l-Phe-Wang resin (100 mg, 0.86 mmol/g,
0.086 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Ala-OH (108 mg, 0.344 mmol) in
the presence of DMT/strychnine/BFꢀ4 (96 mg, 0.172 mmol) and strychnine (12 mg, 0.034 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 28 mg of
10ad as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min) tR 8.11 min, purity > 98%. GC: hydrolyzate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
1
2008, 3 min; tR 18.92 (l-Phe), 4.01 (l-Ala), 3.81 (d-Ala); l/d 8 :92. H-NMR (250 MHz, D2O): 1.34 (d,
J ¼ 7.0, MeCH); 3.08 (d, J ¼ 7.0, CH2CH); 4.45 (q, J ¼ 7.0, MeCH); 4.84 (q, J ¼ 7.0, CH2CH); 6.68 (d, J ¼
7.1, CONH); 7.06 – 7.82 (m, 5 arom. H). LC/MS: 237.39 ([M þ H]þ, C12H17N2O3þ ; calc.237.27).
H-d-Ala-l-Glu-OH (10ae; Table 2, Entry 4). Fmoc-l-Glu(OtBu)-Wang resin (100 mg, 0.81 mmol/g,
0.081 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according to
GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Ala-OH (101 mg, 0.324 mmol) in the
presence of DMT/strychnine/BFꢀ4 (91 mg, 0.162 mmol) and strychnine (11 mg, 0.032 mmol; GP 4). After
deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 24 mg of 10ae
as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 5.25 min, purity > 98%. GC: hydrolysate has
been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; 8.11 min (l-Glu); tR 4.00 (l-Ala), 3.82 (d-Ala); l/d 3 :97. 1H-NMR (250 MHz, D2O): 1.28 (d,
J ¼ 6.5, MeCH)); 2.05 (q, J ¼ 7.2, 2 H, CHꢀCH2CH2); 2.33 (t, J ¼ 6.8, 2 H, CH2ꢀCH2); 4.09 (q, J ¼ 6.5,
MeCH); 4.33 – 4.51 (m, 1 H, CHCOOH). LC/MS: 219.44 ([M þ H]þ, C8H15N2O5þ ; calc. 219.21).
H-d-Ala-l-Pro-OH (10af; Table 2, Entry 5). Fmoc-l-Pro-Wang resin (100 mg, 0.76 mmol/g,
0.076 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Ala-OH (95 mg, 0.304 mmol) in
the presence of DMT/strychnine/BFꢀ4 (85 mg, 0.152 mmol) and strychnine (10 mg, 0.030 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 21 mg of
10af as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 6.11 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 10.81 (l-Pro), 4.19 (l-Ala), 3.89 (d-Ala); l/d 4 :96. 1H-NMR (250 MHz, D2O): 1.37 (d, J ¼
6.0, MeCH); 1.60 – 2.50 (m, CH2ꢀCH2); 3.40 – 3.80 (m, NꢀCH2); 4.18 – 4.28 (m, NꢀCH); 4.43 (q, J ¼ 6.0,
MeCH); 7.08 (br. s, NH). LC/MS: 187.40 ([M þ H]þ, C8H15N2O3þ ; calc. 187.21).
H-l-Phe-Gly-OH (10bb; Table 2, Entry 6). Fmoc-Gly-Wang resin (100 mg, 0.77 mmol/g,
0.077 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Phe-OH (119 mg, 0.308 mmol) in
the presence of DMT/strychnine/BFꢀ4 (86 mg, 0.154 mmol) and strychnine (10 mg, 0.031 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 23 mg of
10bb as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 8.02 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 4.96 (Gly), 18.16 (l-Phe), 17.82 (d-Phe); l/d 90 :10. 1H-NMR (250 MHz, D2O): 3.08 (br. d,

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Helvetica Chimica Acta – Vol. 95 (2012)
J ¼ 6.5, CHꢀCH2Ph); 3.92, 4.00 (d, AB system, J ¼ 14.0, 8.0, NHꢀCH2COO); 4.25 – 4.45 (m,
CHꢀCH2Ph); 7.15 – 7.35 (m, 6 arom. H, NH). LC/MS: 223.65 ([M þ H]þ, C11H15N2O3þ ; calc. 223.25).
H-l-Phe-l-Leu-OH (10bc; Table 2, Entry 7). Fmoc-l-Leu-Wang resin (100 mg, 0.86 mmol/g,
0.086 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Phe-OH (133 mg, 0.344 mmol) in
the presence of DMT/strychnine/BFꢀ4 (96 mg, 0.172 mmol) and strychnine (12 mg, 0.034 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 31 mg of
10bc as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 8.92 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 8.61 (l-Leu), 18.81 (l-Phe), 18.38 (d-Phe); l/d 100 :0. 1H-NMR (250 MHz, D2O): 0.91 (d,
J ¼ 6.0, Me2CH); 1.45 – 1.7 (m, CH2CH); 3.08 (d, J ¼ 7.0, CH2CH); 4.55 (dt, J ¼ 8.0, 4.5, CH2CH); 4.84 (q,
J ¼ 7.0, CH2CH); 6.68 (d, J ¼ 7.0, CONH); 7.06 – 7.52 (m, 5, arom. H). LC/MS: 279.79 ([M þ H]þ,
C15H23N2Oþ3 ; calc. 279.35).
H-l-Phe-l-Ala-OH (10bg; Table 2, Entry 8). Fmoc-l-Ala-Wang resin (100 mg, 0.96 mmol/g,
0.096 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Phe-OH (149 mg, 0.384 mmol) in
the presence of DMT/strychnine/BFꢀ4 (108 mg, 0.192 mmol) and strychnine (13 mg, 0.038 mmol) (GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 32 mg of
10bg as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 7.53 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 4.00 (l-Ala), 18.90 (l-Phe), 18.54 (d-Phe); l/d 98 :2. 1H-NMR (250 MHz, D2O): 1.42 (d,
J ¼ 6.5, MeCH); 3.08 (d, J ¼ 7.0, CH2CH); 4.31 (q, J ¼ 6.5, MeCH); 4.80 (q, J ¼ 7.0, CH2CH); 6.65 (d, J ¼
7.0, CONH); 7.05 – 7.45 (m, 5 arom. H). LC/MS: 237.88 ([M þ H]þ, C12H17N2O3þ ; calc. 237.27).
H-l-Phe-l-Glu-OH (10be; Table 2, Entry 9). Fmoc-l-Glu(OtBu)-Wang resin (100 mg, 0.81 mmol/g,
0.081 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according to
GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Phe-OH (125 mg, 0.324 mmol) in the
presence of DMT/strychnine/BFꢀ4 (91 mg, 0.162 mmol) and strychnine (11 mg, 0.032 mmol; GP 4). After
deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 29 mg of 10be
as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 6.05 min, purity > 98%. GC: hydrolysate has
been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 8.11 (l-Glu), 18.90 (l-Phe), 18.54 (d-Phe); l/d 98 :2. 1H-NMR (250 MHz, D2O): 2.08 (q,
J ¼ 6.1, 2 H, CH2ꢀCH2); 2.35 (t, J ¼ 5.8, 2 H, CH2ꢀCH2); 3.44, 3.19 (dd, J ¼ 2.8, 7.8, CH2CH); 4.16 (t, J ¼
5.9, CHꢀCH2); 4.80 (q, J ¼ 7.0, CH2CH); 6.65 (d, J ¼ 7.0, CONH); 7.05 – 7.45 (m, 5 arom. H). LC/MS:
295.61 ([M þ H]þ, C14H19N2Oþ5 ; calc. 295.31).
H-l-Phe-l-Pro-OH (10bf; Table 2, Entry 10). Fmoc-l-Pro-Wang resin (100 mg, 0.76 mmol/g,
0.076 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Phe-OH (118 mg, 0.304 mmol) in
the presence of DMT/strychnine/BFꢀ4 (85 mg, 0.152 mmol) and strychnine (10 mg, 0.030 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 26 mg of
10bf as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 7.95 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
1
2008, 3 min; tR 10.43 (l-Pro), 18.81 (l-Phe), 18.44 (d-Phe); l/d 96 :4. H-NMR (250 MHz, D2O): 1.40 –
2.88 (m, CH2-CH2); 3.01, 3.16 (d, AB system, J ¼ 14.0, 7.0, PhCH2); 3.30 – 3.60 (m, NꢀCH2); 4.15 – 4.30
(m, CO-N-CH-CO); 4.85 (d, J ¼ 7.0, PhCH2ꢀCH); 6.42 (br. s, NH); 7.00 – 7.49 (m, 5 arom. H). LC/MS:
263.72 ([M þ H]þ, C14H19N2Oþ3 ; calc. 263.31).
H-l-Tyr-Gly-OH (10eb; Table 2, Entry 11). Fmoc-Gly-Wang resin (100 mg, 0.77 mmol/g,
0.077 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Tyr-OH (124 mg, 0.308 mmol) in
the presence of DMT/strychnine/BFꢀ4 (86 mg, 0.154 mmol) and strychnine (10 mg, 0.031 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 24 mg of
10eb as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 6.18 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 5.05 (Gly), 22.86 (l-Tyr), 22.49 (d-Tyr); l/d 89 :11. 1H-NMR (250 MHz, D2O): 3.05, 3.10

Helvetica Chimica Acta – Vol. 95 (2012)
2095
(d, AB system, J ¼ 12.0, 4.0, CHꢀCH2C6H4OH); 3.96 (d, J ¼ 4.0, 2 H, NHꢀCH2COO); 4.65 (q, J ¼ 6.5,
HOC6H4CH2ꢀCH); 5.83 (br., NH); 6.47 (d, J ¼ 8.0, 2 arom. H); 6.99 (d, J ¼ 8.0, 2 arom. H). LC/MS:
239.55 ([M þ H]þ, C11H15N2Oþ4 ; calc. 239.25).
H-l-Tyr-l-Leu-OH (10ec; Table 2, Entry 12). Fmoc-l-Leu-Wang resin (100 mg, 0.86 mmol/g,
0.086 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Tyr-OH (139 mg, 0.344 mmol) in
the presence of DMT/strychnine/BFꢀ4 (96 mg, 0.172 mmol) and strychnine (12 mg, 0.034 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 33 mg of
10ec as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 9.08 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min, 90 – 2008, 48/min,
2008, 3 min; tR 8.29 (l-Leu), 22.76 (l-Tyr), 22.46 (d-Tyr); l/d 100 :0. 1H-NMR (250 MHz, D2O): 0.92 (d,
J ¼ 6.0, Me2CH); 1.42 – 1.72 (m, CH2CH); 3.01, 3.07 (d, AB system, J ¼ 12.0, 4.0, CHꢀCH2C6H4OH); 4.58
(dt, J ¼ 8.0, 4.0, CH2CH); 4.76 (q, J ¼ 6.5, HOC6H4CH2ꢀCH); 5.75 (br., NH); 6.39 (d, J ¼ 8.0, 2 arom. H);
6.96 (d, J ¼ 8.0, 2 arom. H). LC/MS: 295.70 ([M þ H]þ, C15H23N2O4þ ; calc. 295.35).
H-l-Tyr-l-Ala-OH (10eg; Table 2, Entry 13). Fmoc-l-Ala-Wang resin (100 mg, 0.96 mmol/g,
0.096 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Tyr-OH (155 mg, 0.384 mmol) in
the presence of DMT/strychnine/BFꢀ4 (108 mg, 0.192 mmol) and strychnine (13 mg, 0.038 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 32 mg of
10eg as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 8.41 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 4.01 (l-Ala), 22.75 (l-Tyr), 22.46 (d-Tyr); l/d 100 :0. 1H-NMR (250 MHz, D2O): 1.36 (d,
J ¼ 7.0, MeCH); 3.04, 3.11 (d, AB system, J ¼ 12.0, 4.0, CHꢀCH2C6H4OH); 4.44 (qu, J ¼ 7.0, MeCH); 4.75
(q, J ¼ 6.5, HOC6H4CH2ꢀCH); 5.87 (br., NH); 6.46 (d, J ¼ 8.0, 2 arom. H); 6.95 (d, J ¼ 8.0, 2 arom. H).
LC/MS: 253.76 ([M þ H]þ, C12H17N2O4þ ; calc.253.27).
H-l-Tyr-l-Phe-OH (10ed; Table 2, Entry 14). Fmoc-l-Phe-Wang resin (100 mg, 0.86 mmol/g,
0.086 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according
to GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Tyr-OH (139 mg, 0.344 mmol) in
the presence of DMT/strychnine/BFꢀ4 (96 mg, 0.172 mmol) and strychnine (12 mg, 0.034 mmol; GP 4).
After deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 35 mg of
10ed as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 11.23 min, purity > 98%. GC: hydrolysate
has been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 18.99 (l-Phe), 23.34 (l-Tyr), 22.46 (d-Tyr); l/d 98 :2. 1H-NMR (250 MHz, D2O): 3.01, 3.07
(d, AB system, J ¼ 12.0, 4.0, CHꢀCH2C6H4OH); 3.11, 315 (d, AB system, J ¼ 14.0, 5.0, CHꢀCH2Ph); 4.76
(q, J ¼ 6.5, HOC6H4CH2ꢀCH); 4.88 (q, J ¼ 6.5, PhCH2ꢀCH); 5.77 (br., NH); 6.36 (d, J ¼ 8.0, 2 arom. H);
6.90 (d, J ¼ 8.0, 2 arom. H); 7.06 – 7.35 (m, 5 arom. H). LC/MS: 329.81 ([M þ H]þ, C18H21N2O4þ ; calc.
329.37).
H-l-Tyr-l-Glu-OH (10ee; Table 2, Entry 15). Fmoc-l-Glu(OtBu)-Wang resin (100 mg, 0.81 mmol/g,
0.081 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according to
GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Tyr-OH (131 mg, 0.324 mmol) in the
presence of DMT/strychnine/BFꢀ4 (91 mg, 0.162 mmol) and strychnine (11 mg, 0.032 mmol; GP 4). After
deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 30 mg of 10ee
as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 7.15 min, purity > 98%. GC: hydrolysate has
been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 8.10 (l-Glu), 23.34 (l-Tyr), 22.46 (d-Tyr); l/d 100 :0. 1H-NMR (250 MHz, D2O): 2.04 (q,
J ¼ 6.8, 2 H, CH2ꢀCH2); 2.35 (t, J ¼ 5.7, 2 H, CH2ꢀCH2); 3.04, 3.11 (d, AB system, J ¼ 12.0, 4.0,
CHꢀCH2C6H4OH); 4.14 (t, J ¼ 6.9, 1 H, CH2CH); 4.75 (q, J ¼ 6.5, HOC6H4CH2ꢀCH); 5.87 (br., NH);
6.46 (d, J ¼ 8.0, 2 arom. H); 6.95 (d, J ¼ 8.0, 2 arom. H). LC/MS: 311.71 ([M þ H]þ, C14H19N2O6þ ; calc.
311.31).
weiterH-l-Tyr-l-Pro-OH (10ef; Table 2, Entry 16). Fmoc-l-Pro-Wang resin (100 mg, 0.76 mmol/g,
0.076 mmol) was pre-swollen in CH2Cl2 (5 ml) for 1 h, and then Fmoc group was removed according to
GP 2. Subsequently, the peptide chain was elongated with Fmoc-dl-Tyr-OH (123 mg, 0.304 mmol) in the
presence of DMT/strychnine/BFꢀ4 (85 mg, 0.152 mmol) and strychnine (10 mg, 0.030 mmol; GP 4). After

2096
Helvetica Chimica Acta – Vol. 95 (2012)
deprotection (GP 2), dipeptide was cleaved from the resin (GP 3) and lyophilized to give 26 mg of 10ef
as a TFA salt. Anal. RP-HPLC (0 – 10% B in 20 min): tR 8.28 min, purity > 98%. GC: hydrolysate has
been derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min;
2008, 3 min; tR 10.43 (l-Pro), 23.33 (l-Tyr), 22.46 (d-Tyr); l/d 99 :1. 1H-NMR (250 MHz, D2O): 1.40 – 2.88
(m, CH2ꢀCH2); 3.04, 3.11 (d, AB system, J ¼ 12.0, 4.0, CHꢀCH2C6H4OH); 4.14 (t, J ¼ 6.9, CH2CH);
3.30 – 3.60 (m, NꢀCH2); 4.15 – 4.30 (m, CO-N-CH-CO); 4.75 (q, J ¼ 6.5, HOC6H4CH2ꢀCH); 6.46 (d, J ¼
8.0, 2 arom. H); 6.95 (d, J ¼ 8.0, 2 arom. H). LC/MS: 279.70 ([M þ H]þ, C14H19N2O4þ ; calc. 279.31).
Ac-l-Lys(Ac)-d-Ala-d-Ala-OH (11): Anchoring Fmoc-d-Ala-OH to Wang Resin (Table 3, Entry 1).
To the vigorously stirred soln. of DMT/strychnine/BFꢀ4 (6) (0.308 g, 0.55 mmol) in MeCN (5 ml), cooled
to 08, strychnine (37 mg, 0.11 mmol) was added, and after 5 min the soln. was treated with the
appropriate racemic carboxylic acid (0.242 g, 1.1 mmol). The stirring was continued for additional
15 min, then the soln. was added to the pre-swollen (in CH2Cl2 for 1 h) Wang resin (0.250 g, 1.1 mmol/g,
0.275 mmol) treated with stoichiometric amount of EtNiPr2 (50 ml, 0.275 mmol) and cat. amount of
DMAP (5 mg), and the suspension was shaked for 10 h. The resin was then filtered, washed with CH2Cl2
(4 ml, 6 ꢁ 1 min), and dried under h.v. overnight. The level of alanine-residue attachment to Wang resin
[71] was estimated as 0.95 mmol/g of the modified resin. In the next step, the Fmoc-Ala-Wang resin was
covered with DMF (5 ml), and unreacted OH groups were capped using Ac2O (0.281 g, 2.75 mmol) and
DMAP (3 mg, 0.027 mmol) dissolved in DMF (0.5 ml) for 1 – 2 h under Ar bubbling. The resin was then
washed with DMF (5 ml, 5 ꢁ 1 min) and CH2Cl2 (5 ml, 5 ꢁ 1 min), and then the Fmoc group was
removed according to GP 2. A small amount of Ala-Wang resin (50 mg) was treated with TFA/Et3SiH/
H2O 9.5 :2.5 :2.5 (5 ml). Cleavage was performed during 2 h, then the resin was filtered off, and the
filtrate was evaporated. The solid residue was derivatizated according to GP 5; chromatography
conditions: temp. 908, 4 min; 90 – 2008, 48/min; 2008, 3 min; tR 4.00 (l-Ala), 3.81 (d-Ala); d/l 90 :10.
Incorporation of Second Residue of d-Ala to d-Ala-Wang Resin (Table 3, Entry 2). Subsequently, the
peptide chain was elongated with Fmoc-dl-Ala-OH (0.236 g, 0.760 mmol) in the presence of DMT/
strychnine/BFꢀ4 (0.213 g, 0.38 mmol) and strychnine (25 mg, 0.076 mmol; GP 4). After deprotection
(GP 2), 50 mg of Ala-Ala-Wang resin was treated with TFA/Et3SiH/H2O 9.5 :2.5 :2.5 (5 ml). Cleavage
was performed during 2 h, then the resin was filtered off, and the filtrate was evaporated. Solid residue
was dissolved in H2O (5 ml) and lyophilized to give 11 mg of d-Ala-d-Ala as a TFA salt. Anal. RP-HPLC
(0 – 10% B in 20 min): tR 6.11 min, purity > 98%. GC: hydrolysate has been derivatized according to
GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min, 2008; 3 min; tR 4.01 (l-Ala), 3.81
(d-Ala); d/l 95 :5. LC/MS: 161.73 ([M þ H]þ, C6H13N2O3þ ; calc.161.17).
Incorporation of Lys Residue to d-Ala-d-Ala-Wang Resin (Table 3, Entry 3). Fmoc-Lys(Fmoc)-OH
(0.284 mmol), EtN(i-Pr)2 (102 ml, 0.568 mmol), HOBt (0.284 mmol), and DMT/NMM/BFꢀ4 (2; 93 mg,
0.284 mmol) as a coupling reagent were dissolved in DMF (5 ml) and added to d-Ala-d-Ala-Wang resin
(150 mg, 0.95 mmol/g, 0.142 mmol) and shaken for 30 min. The completion of the reaction was confirmed
by TNBS test. After Fmoc deprotection (GP 2), tripeptide was as covered with DMF (3 ml), and free
amino groups were acetylated using Ac2O (0.290 g, 2.84 mmol.) and DMAP (3 mg, 0.028 mmol)
dissolved in DMF (0.3 ml) for 2 h under Ar bubbling. The resin was then washed with DMF (5 ml, 5 ꢁ
1 min) and CH2Cl2 (5 ml, 5 ꢁ 1 min). Tripeptide was cleaved from the resin (GP 3) and lyophilized to
give 45 mg of 11.
Anal. RP-HPLC (0 – 34% B in 20 min). tR 10.11 min, purity > 97%. GC: hydrolysate has been
derivatized according to GP 5; chromatography conditions: temp. 908, 4 min; 90 – 2008, 48/min; 2008,
3 min; tR 4.01 (l-Ala), 3.81 (d-Ala); d/l 95 :5; 11.03 (l-Lys), 11.28 (d-Lys); d/l 0 :100. 1H-NMR
(250 MHz, CD3OD): 1.25 (q, J ¼ 4.8, 2 H, CH2ꢀCH2ꢀCH2); 1.43 (d, J ¼ 6.9, MeCH); 1.48 (d, J ¼ 6.9,
MeCH); 1.55 (q, J ¼ 4.7, 2 H, CH2ꢀCH2ꢀCH2); 1.80 (q, J ¼ 6.1, 2 H, CH2ꢀCH2ꢀCH2); 1.85 (s, 2 MeCO);
3.01, 3.06 (dt, J ¼ 2.4, 5.9, NHCH2); 4.11 (q, J ¼ 6.9, MeCH); 4.41 (t, J ¼ 5.5, CHꢀCH2); 4.53 (q, J ¼ 6.9,
MeCH); 7.2 – 7.9 (m, 4 NH). LC/MS: 372.53 (Mþ, C16H28N4O6þ ; calc. 372.42).

Helvetica Chimica Acta – Vol. 95 (2012)
2097
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Received August 8, 2012