Preparation of N-Fmoc-protected (S)-5-amino-4,4-difluoro-7-methyloctanoic acid, a possible dipeptide isostere

Article abstract of DOI:10.1002/hlca.200900215

The title compound 1 was prepared from l-leucine. The key steps include a Grignard addition to Bn₂-leucinal, a CO/CF₂ replacement with Et₂NSF₃ (DAST) and use of a Ph group as synthetic equivalent of a COOH group

Full text of DOI:10.1002/hlca.200900215

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Helvetica Chimica Acta – Vol. 92 (2009)
Preparation of N-Fmoc-Protected (S)-5-Amino-4,4-difluoro-7-
methyloctanoic Acid, a Possible Dipeptide Isostere
by Gildas Deniau1) and Dieter Seebach*
Laboratorium fꢀr Organische Chemie, Departement fꢀr Chemie und Angewandte Biowissenschaften,
ETH-Zꢀrich, Hçnggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(phone: þ 41-44-632-2990; fax: þ 41-44-632-1144; e-mail: seebach@org.chem.ethz.ch)
The title compound 1 was prepared from l-leucine. The key steps include a Grignard addition to
Bn2-leucinal, a CO/CF2 replacement with Et2NSF3 (DAST) and use of a Ph group as synthetic equivalent
of a COOH group. The difluoro-d-amino acid 1 was incorporated into a peptide 8; tests with various
proteases showed no inhibition by this particular peptide.
Statine B [1], homostatine D [2], and ketomethylene analogs C [3] have been
widely used as building blocks in peptides for inhibition of aspartic proteinases [4],
notably of renin [5], and of aminopeptidases [6]. While the statines B are g-amino-acid
derivatives (providing five chain atoms), the 5-amino-4-oxo- and the 5-amino-4-
hydroxy-carboxylic acid derivatives C and D contain six chain atoms, just like a
dipeptide segment A of a ꢁnormal peptideꢂ (ꢁdipeptide isosteresꢂ).
In spite of the fact that ꢁOrganic Fluorine Hardly Ever Accepts Hydrogen Bondsꢂ,
[7] the literature is full of reports2) describing compounds containing CꢀF groups in
positions where corresponding model compounds contain CꢀOH groups, with
comparisons of physiological properties of the two types of compounds [8][9]. To
provide another test case, we have prepared the N-protected difluoro amino acid 1 and
incorporated it into a peptide for biological tests.
The starting point of the synthesis (Scheme 1) was (S)-2-(dibenzylamino)-4-
methylpentanal (2), an aldehyde derived from l-leucine [10]. A Ph ring was used as a
hidden COOH moiety [11][12] to avoid cyclizations of intermediates with formation of
a lactone or lactam ring. Addition of 2-phenylethyl Grignard reagent to the aldehyde
group gave a readily separated ca. 12 :1 mixture of two crystalline diastereoisomeric
amino alcohols (89% yield). By analogy with the literature [10], the major
diastereoisomer 3 is assigned (3R,4S)-configuration (ꢁnon-chelation-controlledꢂ ap-
proach of the nucleophile from the Re-face of the C¼O group, relative topicity ul).
Swern oxidation [13] of the amino alcohol 3 gave the amino ketone 4 as an oil in
91% yield. Treatment of the ketone with eightfold excess of Et2NSF3 (DAST) [14]
(CH2Cl2, ꢀ 5 to þ 208, 4 d) provided the difluoro amine 5 in 55% yield. The loss of one
1
)
Postdoctoral Fellow, ETH-Zꢀrich (2007 – 2009), financially supported by the Swiss National Science
Foundation (Project No. 200020-117586) and by Novartis Pharma AG.
Especially in the patent literature describing pharmaceutical structure – activity relationships.
2
)
ꢃ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

Helvetica Chimica Acta – Vol. 92 (2009)
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Scheme 1. Preparation of the d-Amino Acid Derivative 1
of the N-bound Bn groups in this process might be the result of the sequence of steps
outlined in Scheme 2.
The remaining steps are functional-group manipulations: Bn/(tert-butoxy)carbonyl
(Boc) exchange (H2/Pd-C, Boc2O ! 87% 6), oxidative degradation of the Ph ring to a

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Helvetica Chimica Acta – Vol. 92 (2009)
Scheme 2. Possible Mode of Loss of a Benzyl Group in the Process of Replacement of CO by CF2 (4 ! 5)
with DAST (Et2NSF3). Compare with the reports on OH/F-retention substitution in a-hydroxy-b-amino
acid derivatives [15] and related works [16].
COOH group (RuCl3/NaIO4 ! 7) and direct Boc/(9H-fluoren-9-yl)methoxycarbonyl
(Fmoc) interchange (CF3CO2H, then Fmoc-OSu/Na2CO3 ! 1, 55% overall from 6).
Both the Boc- and the Fmoc-protected amino acids 7 and 1, respectively, consisted of
mixtures of rotamers as evident from temperature-dependent NMR spectra in
(D6)DMSO (see Exper. Part).
The 5-(Fmoc-amino)-4,4-difluoro-7-methyloctanoic acid 1, thus obtained, has a
sharp melting point and is optically active ([a]D ¼ ꢀ16.4), like all the intermediates of
the sequence of reactions leading to 1. Although we did not determine the enantiomer
purity of any of the compounds 1 – 7, for instance by HPLC on chiral column materials
or by NMR techniques, we have no reason to assume that there has been partial
racemization on the way from l-leucine to the acid 1.
To see whether a peptide containing the amino-difluoro-acyl moiety of 1 could
possibly function as a dipeptide isostere (cf. ketomethylene in C with the CF2ꢀCH2
segment in 1) we have, so far, prepared only one peptide analog. We chose the
octapeptide E, which, apart from replacement of the C-terminal His by a Tyr residue, is
the 6 – 13 sequence of angiotensinogen, the substrate of human renin. Peptides of this
kind had first been prepared in 1973, in the course of a search for renin inhibitors [17].
Thus, the peptide 8, containing seven amino acids, was synthesized by the ꢁFmoc
strategyꢂ [18] on Wang resin [19]. The crude product solution obtained by cleavage
from the resin was lyophilized, and the residue purified (> 98%) by preparative HPL
chromatography, and the pure peptide 8 was identified by NMR spectroscopy and high-
resolution mass spectrometry.
Tests with enzymes hBAC, hCathepsin, hPepsin, and hRenin3) showed that there is
no cleavage and no inhibition by peptide 8. The use of the d-amino-acid moiety of 1 in
other peptides, especially those in which the ketomethylene replacement C was
successful, will provide a further test of the viability of the CO/CF2 replacement.
3
)
We thank Drs. H. Sellner and O. Dreier of Novartis Pharma AG, Basel, for carrying out these
experiments.

Helvetica Chimica Acta – Vol. 92 (2009)
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Furthermore, g,g-difluoro-acid analogs of 1 with an additional side chain in the a-
position (cf. homostatines D, R=H) should be targets of future investigations.
Experimental Part
General. Abbreviations. DMAP: 4-(dimethylamino)pyridine, Bn: benzyl, DAST: Et2NSF3, Fmoc-
OSu: N-{[(9H-fluoren-9-ylmethoxy)carbonyl]oxy}succinimide, HATU: O-(7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate, TFA: CF3COOH, TNBS: 2,4,6-trinitrobenzenesul-
fonic acid, MeIm: 1-methyl-1H-imidazole, MSNT: 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazol,
HPLC: high-performance liquid chromatography, MALDI: matrix-assisted laser-desorption ionization.
Materials and Methods. All reagents were of synthetic grade and were used without further
purification unless otherwise stated. Dry THF was distilled from Na and benzophenone; dry CH2Cl2 was
distilled from CaH2. All moisture-sensitive reactions were carried out under a positive pressure of N2 in
oven-dried glassware (1408). Org. extracts were dried over MgSO4. TLC: Merck TLC silica gel 60 F254
aluminum plates; visualization by inspection under UV light (254 nm) or by the use of KMnO4 stain, Mo-
based stain, bromocresol green stain, or ninhydrin spray. Column chromatography (CC): silica gel 60
(40 – 63 m) from Fluka. Wang resin was purchased from Novabiochem, amino acids were purchased from
Fluka. Reversed-phase (RP) HPLC: Merck/Hitachi HPLC system (LaChrom, pump type L-7150, UV-
detector L-7400, interface D-7000, HPLC-manager D-7000). Anal. HPLC: Macherey-Nagel C18 column
(Nucleosil 100-5 C18 (250 ꢁ 4 mm)) using a gradient of solvent A (MeCN) and B (0.1% TFA in H2O) at a
flow rate of 1 ml/min. Prep. HPLC: Macherey-Nagel C18 column (Nucleosil 100 – 5 C18 (250 ꢁ 21 mm))
using a gradient of solvent A (MeCN) and B (0.1% TFA in H2O) at a flow rate of 10 ml/min.
Lyophilization was realized using a Hetosicc cooling condenser with high-vacuum pump to obtain
peptides as their TFA salts. M.p.: Bꢀchi 510 melting-point apparatus; uncorrected. Optical rotations:

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Helvetica Chimica Acta – Vol. 92 (2009)
Perkin-Elmer 241 polarimeter (10 cm, 1-ml cell), [a]D values are determined at 589 nm and given in 10ꢀ1
deg cm2 gꢀ1. IR Spectra: neat (unless otherwise stated) on a Perkin-Elmer precisely Universal ATR
Sampling Accessory; in cmꢀ1. NMR Spectra: either Bruker AV-300 (1H: 300.06 MHz, 13C: 75.45 MHz,
19F: 282.34 MHz), or AMX-400 (1H: 400.95 MHz, 13C: 100.12 MHz), or AV-600 (1H: 600.13 MHz, 13C:
150.91 MHz, 19F: 376.33 MHz); chemical shifts d are reported in ppm relative to internal standard Me4Si
1
1
for H and 13C, and to external standard CFCl3 for 19F; coupling constants J in Hz; H-, 13C-, and 19F-
spectroscopic data assigned on a routine basis by a combination of 1D and 2D experiments (COSY,
HSCQ, HMBC, NOESY). High-resolution (HR) MS: IonSpec Ultima 4.7 T FT Ion Cyclotron
Resonance (ICR, HR-MALDI-MS, in a 2,5-dihydrobenzoic acid matrix) mass spectrometer in m/z (%
of basis peak). Elemental analyses were performed by the Microanalytical Laboratory of the
Laboratorium fꢀr Organische Chemie, ETH-Zꢀrich.
Preparation of Compound 1. (3R,4S)-4-(Dibenzylamino)-6-methyl-1-phenylheptan-3-ol (3). A soln.
of (2-bromoethyl)benzene (4.06 ml, 30 mmol, 1.8 equiv.) in dry THF (30 ml) was added dropwise at r.t.
into a round-bottom flask filled with N2 and containing Mg turnings (778 mg, 32 mmol, 1.9 equiv.), one
crystal of I2, and dry THF (50 ml). The mixture was stirred at r.t. for 2 h, and a soln. of N,N-dibenzyl-l-
leucinal (2) (prepared according to the literature procedure [10b]; 5 g, 17 mmol) in dry THF (20 ml) was
added dropwise over 10 min. The mixture was stirred for an additional 2 h, and the reaction was
quenched by the addition of sat. NH4Cl soln. (80 ml). The product was extracted with AcOEt, and the
combined org. phases were dried (MgSO4) and evaporated under reduced pressure. The residue was
purified by CC (hexane/AcOEt 15 :1) to give the minor diastereoisomer (3S,4S)-4-(dibenzylamino)-6-
methyl-1-phenylheptan-3-ol as a colorless crystalline solid, which eluted first (450 mg, 7% yield). The
main product 3 was obtained as a colorless oil which slowly crystallized (5.58 g, 82% yield). M.p. 73 – 748
(hexane/CH2Cl2). Rf (hexane/AcOEt 10 :1) 0.31. [a]2D0 ¼ ꢀ3.1 (c ¼ 1, CHCl3). IR: 3456 (br.), 3027w,
2951m, 1603w, 1495s, 1453s, 1365w, 1065m, 1028m, 746s, 698s. 1H-NMR (400 MHz, CDCl3): 0.73 (d, J ¼
6.5, Me); 0.88 (d, J ¼ 6.5, Me); 1.23 (td, J ¼ 6.9, 13.8, 1 H, CH2); 1.57 (td, J ¼ 6.9, 13.8, 1 H, CH2); 1.63 –
180 (m, CH2, Me2CH); 2.31 (br. s, OH); 2.56 (ddd, J ¼ 7.2, 9.2, 13.8, 1 H, PhCH2); 2.74 (dt, J ¼ 4.0, 7.0,
CHN); 2.87 (ddd, J ¼ 5.0, 9.0, 14.2, 1 H, PhCH2); 3.63 (s, 2 PhCH2N); 3.73 (td, J ¼ 3.4, 9.8, CHOH);
7.14 – 7.31 (m, 15 arom. H); 13C-NMR (75 MHz, CDCl3): 22.9 (Me); 23.0 (Me); 24.8 (CH); 33.1 (CH2);
34.5 (CH2); 36.3 (CH2); 55.2 (2 PhCH2N); 58.6 (CHN); 69.9 (CHOH); 125.7; 127.0 (2 C); 128.2 (4 C);
128.3 (2 C); 128.4 (2 C); 128.9 (4 C); 139.9 (2 C); 142.0. ESI-MS: 402.27942 ([M þ H]þ, C28H36NOþ; calc.
402.27914 (þ0.7 ppm)).
(S)-4-(Dibenzylamino)-6-methyl-1-phenylheptan-3-one (4). To a stirred soln. of oxalyl chloride
(0.805 ml, 9.35 mmol, 1.5 equiv.) in dry CH2Cl2 (20 ml) cooled at ꢀ 788 under N2 was added dropwise
DMSO (1.33 ml, 18.7 mmol, 3 equiv.). After stirring for 15 min, a soln. of 3 (2.5 g, 6.23 mmol) in dry
CH2Cl2 (15 ml) was added dropwise at ꢀ 788. The mixture was stirred at this temp. for 30 min, and Et3N
(2.5 ml) was added. The mixture was then allowed to slowly warm to r.t. over 2 h, and the reaction was
quenched with H2O (20 ml). The product was extracted with CH2Cl2, and the combined org. phases were
dried (MgSO4) and evaporated under reduced pressure. The residue was purified by CC (hexane/AcOEt
20 :1) to yield 4 (2.28 g, 91%). Colorless oil. [a]2D0 ¼ ꢀ91.6 (c ¼ 1, CHCl3). IR: 3027w, 2954m, 1713s,
1603w, 1495s, 1453s, 1368m, 1071w, 1028w, 746s, 698s. 1H-NMR (300 MHz, CDCl3): 0.71 (d, J ¼ 6.4, Me);
0.80 (d, J ¼ 6.4, Me); 1.34 – 1.48 (m, CH, 1 H of CH2); 1.70 (ddd, J ¼ 5.1, 8.8, 12.9, 1 H, CH2); 2.62 – 2.70
(m, 1 H, CH2CO); 2.74 – 2.92 (m, 1 H of CH2CO, PhCH2); 3.23 (dd, J ¼ 4.2, 8.8, CHN); 3.50 (d, J ¼ 13.7,
2 ꢁ 1 H, PhCH2N); 3.67 (d, J ¼ 13.7, 2 ꢁ 1 H, PhCH2N); 7.08 – 7.31 (m, 15 arom. H). 13C-NMR (75 MHz,
CDCl3): 22.5 (Me); 23.2 (Me); 25.5 (Me2CH); 30.0 (PhCH2); 32.2 (CHCH2); 42.4 (CH2CO); 54.6 (2 ꢁ
PhCH2N); 64.2 (CHN); 126.0; 127.2 (2 C); 128.3 (4 C); 128.4 (2 C); 128.5 (2 C); 129.0 (4 C); 139.7 (2 C);
141.3; 210.7 (CO). HR-MALDI-MS: 400.2635 ([M þ H]þ, C28H34NOþ; calc. 400.2635 (þ0.0 ppm)).
(S)-Benzyl-3,3-difluoro-6-methyl-1-phenylheptan-4-amine (5). To a soln. of 4 (2 g, 5 mmol) in dry
CH2Cl2 (10 ml) stirred at ꢀ 58 under Ar was added dropwise DAST (4.9 ml, 40 mmol, 8 equiv.). The
mixture was allowed to slowly warm to r.t. and stirred for 4 d. The mixture was then cooled to 08, and the
reaction was quenched by the addition of sat. NaHCO3 soln. (10 ml). The product was extracted with
CHCl3, the combined org. layers were evaporated under reduced pressure, and the residue was purified
by CC (hexane/AcOEt 40 :1) to give 5 (930 mg, 55% yield). Yellowish oil. [a]2D0 ¼ ꢀ30.8 (c ¼ 1, CHCl3).
IR: 3028w, 2956s, 1496m, 1454s, 1379m, 1187w, 1123m, 1044m, 936s, 744s, 698s. 1H-NMR (300 MHz,

Helvetica Chimica Acta – Vol. 92 (2009)
1725
CDCl3): 0.81 (d, J ¼ 6.5, Me); 0.91 (d, J ¼ 6.6, Me); 1.16 (br. s, NH); 1.30 (ddd, J ¼ 4.8, 9.6, 13.8, 1 H,
CH2); 1.40 (ddd, J ¼ 3.5, 9.4, 13.4, 1 H, CH2); 1.69 – 1.79 (m, Me2CH); 2.05 – 2.21 (m, 1 H, CH2CF2);
2.24 – 2.40 (m, 1 H, CH2CF2); 2.79 – 2.94 (m, CHN, PhCH2); 3.82 (d, J ¼ 13.0, 1 H, PhCH2N); 3.93 (d,
J ¼ 13.0, 1 H, PhCH2N); 7.16 – 7.33 (m, 10 arom. H). 13C-NMR (75 MHz, CDCl3): 21.7 (Me); 23.8 (Me);
25.0 (Me2CH); 27.9 (t, J ¼ 5.0, PhCH2); 35.0 (t, J ¼ 24.7, CH2CF2); 39.2 (t, J ¼ 3.4, CH2); 53.0
(PhCH2N); 59.1 (t, J ¼ 25.4, CHN); 126.1; 126.47 (t, J ¼ 245.7, CF2); 127.1; 128.4 (2 C); 128.5 (4 C); 128.6
(2 C); 140.7; 141.3. 19F-NMR (375 MHz, CDCl3): ꢀ 104.67 (tdd, J ¼ 10.0, 26.7, 245.0, 1 F); ꢀ 106.75 (tdd,
J ¼ 9.8, 26.5, 245.0, 1 F). ESI-MS: 332.2186 ([M þ H]þ, C21H28F2Nþ; calc. 332.2184 (þ0.45 ppm)).
(S)-N-[(tert-Butoxy)carbonyl]-3,3-difluoro-6-methyl-1-phenylheptan-4-amine (6). To a soln. of 5
(565 mg, 1.7 mmol) in MeOH (15 ml) was added Pd/C 10% (160 mg). The mixture was stirred at r.t. for
2 h under atmospheric pressure of H2. Boc2O (745 mg, 3.4 mmol, 2 equiv.) was added, and the black
suspension was stirred for 24 h under H2. The mixture was filtered over Celiteꢄ to remove Pd/C, and the
solvent was evaporated under reduced pressure. The residue was taken in Et2O (20 ml), and H2O (4 ml)
was added. The org. phase was separated, dried (MgSO4), and evaporated under reduced pressure. The
residue was purified by CC (100% hexane, then hexane/AcOEt 30 :1) to give 6 (505 mg, 87% yield).
Colorless crystalline solid. M.p. 76 – 778 (from hexane/AcOEt). [a]2D0 ¼ ꢀ18.9 (c ¼ 1, CHCl3). IR: 3364m,
2961s, 1701s, 1506s, 1367s, 1254m, 1166s, 1044s, 940m, 747m, 700s. 1H-NMR (300 MHz, CDCl3): 0.94 (d,
J ¼ 6.5, Me); 0.96 (d, J ¼ 6.6, Me); 1.45 (s, 3 Me); 1.36 – 1.60 (m, CH2); 1.64 – 1.74 (m, Me2CH); 2.09 –
2.25 (m, CH2CF2); 2.73 – 2.95 (m, PhCH2); 4.06 (m, CHN); 4.47 (d, J ¼ 10.2, NH); 7.17 – 7.30 (m, 5 arom.
H). 13C-NMR (100 MHz, CDCl3): 21.3 (Me); 23.7 (Me); 24.5 (Me2CH); 28.1 (t, J ¼ 4.9, PhCH2); 28.3
(3 Me); 36.1 (t, J ¼ 24.5, CH2CF2); 37.0 (CH2); 52.0 (dd, J ¼ 24.4, 30.4, CHN); 79.9 (CMe3); 123.9 (t, J ¼
246.2, CF2); 126.1; 128.4 (2 C); 128.5 (2 C); 140.8; 155.6 (CO). 19F-NMR (375 MHz, CDCl3): ꢀ 108.73
(ddd, J ¼ 6.5, 18.7, 246.0, 1 F); ꢀ 111.41 (ddd, J ¼ 18.8, 31.1, 246.2, 1 F). ESI-MS: 364.20567 ([M þ Na]þ,
C19H29F2NNaOþ2 ; calc. 364.20586 (ꢀ0.52 ppm)). Anal. calc. for C19H29NO2F2: C 66.84, H 8.56, N 4.10;
found: C 66.57, H 8.46, N 4.11.
(S)-5-{[(tert-Butoxy)carbonyl]amino}-4,4-difluoro-7-methyloctanoic Acid (7). Compound 6
(300 mg, 0.88 mmol) was dissolved in the biphasic solvent system CCl4/MeCN/H2O (8 :8, 10 ml), and
NaIO4 (3.385 g, 15.8 mmol, 18 equiv.) was added. The mixture was then treated with RuCl3 · x H2O (36%
Ru; 15 mg, 0.05 mmol, 6 mol-%), and the mixture was stirred vigorously at 258 in a water bath for 2 d.
The mixture was then filtered through a large Celiteꢄ pad, and the solids were washed with AcOEt. The
filtrate was concentrated to dryness to give a brownish residue that was used directly for the next
step. 1H-NMR (300 MHz, CDCl3): 0.92 (d, J ¼ 6.5, Me); 0.95 (d, J ¼ 6.5, Me); 1.37 – 1.54 (m, CH2); 1.44
(s, 9 H); 1.61 – 1.75 (m, Me2CH); 2.14 – 2.32 (m, CH2CF2); 2.52 – 2.73 (m, CH2CO2); 3.81 – 4.06 (m,
CHN); 4.49 (d, J ¼ 10.2, NH, major rotamer). 19F-NMR (285 MHz, CDCl3): ꢀ 108.09 (dm, J ¼ 249.0,
minor rotamer); ꢀ 109.13 (dm, J ¼ 248.8, 1 F); ꢀ 111.84 (dm, J ¼ 249.2, minor rotamer); ꢀ 112.92 (dm,
J ¼ 247.8, 1 F).
(S)-5-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-4,4-difluoro-7-methyloctanoic Acid (1).
Crude 7 was treated at r.t. with TFA (8 ml), and the soln. was stirred at r.t. for 1 h. CHCl3 was added,
and the solvents were evaporated under reduced pressure. Final traces of TFA were removed under high
vacuum, and the resulting residue was dissolved in 0.2m Na2CO3 (17 ml, 4 equiv.). A soln. of Fmoc-OSu
(445 mg, 1.3 mmol, 1.5 equiv.) in acetone (8 ml) was then added dropwise, and the mixture was stirred at
r.t. for 6 h. Acetone was evaporated under reduced pressure, the residual soln. was extracted with Et2O
(3 ꢁ 10 ml), and the aq. phase was acidified to pH 2 with 6n HCl before being extracted with AcOEt
(3 ꢁ 10 ml). The org. extracts were combined, dried (MgSO4), and evaporated. Purification by CC
(hexane/AcOEt 3 :1, 0.5% AcOH) afforded 1 (209 mg, 55% yield). Colorless solid. M.p. 130 – 1328
(hexane/AcOEt). [a]2D0 ¼ ꢀ16.4 (c ¼ 1, CHCl3). IR: 2959m, 1713s, 1520m, 1450m, 1260m, 1216s, 1114w,
1056m, 947m, 757s. 1H-NMR (400 MHz, (D6)DMSO)4): 0.45 (d, J ¼ 6.5, Me rotamer 10%); 0.74 (d, J ¼
6.5, Me rotamer 10%); 0.80 (d, J ¼ 6.6, Me); 0.88 (d, J ¼ 6.6, Me); 1.22 – 1.32 (m, 1 H, CH2); 1.46 – 1.57
(m, Me2CH, 1 H of CH2); 2.02 – 2.17 (m, CH2CF2); 2.30 – 2.45 (m, CH2CO2); 3.77 – 3.90 (m, CHN); 4.21
(t, J ¼ 6.9, CH of Fmoc); 4.37 (d, J ¼ 6.9, CH2O); 7.28 – 7.33 (m, 2 arom. H); 7.38 – 7.42 (m, 2 arom. H);
4
)
NMR Measurement at 608 showed disappearance of the minor rotamer signals with concomitant
broadening of the signals of the major rotamer.

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Helvetica Chimica Acta – Vol. 92 (2009)
7.60 (d, J ¼ 9.4, NH); 7.69 (dd, J ¼ 4.2, 7.1, 2 arom. H); 7.87 (d, J ¼ 7.5, 2 arom. H); 12.05 (br. s, CO2H).
13C-NMR (100 MHz, (D6)DMSO): 21.3 (Me); 23.7 (Me); 24.0 (Me2CH); 26.5 (CH2CO2H); 28.3 (t, J ¼
24.7, CH2CF2); 35.6 (CH2); 46.8 (CH of Fmoc); 52.7 (t, J ¼ 27.6, CHN); 65.4 (CH2O); 120.4 (2 C); 124.4
(t, J ¼ 247.8, CF2); 125.4 (2 C); 127.2 (2 C); 127.9 (2 C); 141.0 (2 C); 144.2 (2 C); 156.8 (CO); 173.5
(CO2H). 19F-NMR (375 MHz, (D6)DMSO): ꢀ 106.63 (ddt, J ¼ 241.8, 18.3, 8.2, 1 F); ꢀ 109.03 (dm, J ¼
241.8, 1 F). HR-MALDI-MS: 454.1800 ([M þ Na]þ, C24H27F2NNaO4þ ; calc. 454.18004 (1.32 ppm)).
Preparation of 2 · TFA. H-(S)-His-(S)-Pro-(S)-Phe-(S)-His-(S)-3,3-difluoro-d-h(F2)hhLeu-(S)-Ile-
(S)-Tyr-OH (8). Anchorage of N-Fmoc-Protected Amino Acid on Wang Resin. Esterification of the N-
Fmoc-Tyr(OtBu)-OH with Wang resin was performed by the MSNT/MeIm method [20]. The resin
(75 mg, 1.1 mmol/g, 100 – 200 mesh) was placed in a dry manual reactor and swollen in CH2Cl2 (3 ml) for
30 min. A soln. of N-Fmoc-Tyr(OtBu)-OH (200 mg, 0.44 mmol, 5 equiv.), MeIm (26 ml, 0.33 mmol, 3.75
equiv.), and MSNT (130 mg, 0.44 mmol, 5 equiv.) in CH2Cl2 (3 ml) was then added to the swollen resin
under N2, and the suspension was mixed by N2 bubbling for 4 h. The resin was filtered, washed with
CH2Cl2, and dried under vacuum for 14 h. The resin substitution was determined by measuring the
absorbance of the dibenzofulvene – piperidine adduct [18b] and was found to be 0.93 mmol/g (85%).
Capping Procedure. The peptide – resin was taken in a soln. of Ac2O (66 ml, 10 equiv.) and DMAP
(0.2 equiv.) in CH2Cl2 (3 ml), and the suspension was mixed by N2 bubbling for 2 h. The resin was then
washed by CH2Cl2 (5 ꢁ 5 ml, 1 min).
Deprotection of N-Fmoc-Protected Amino Acids on Wang Resin (GP 1). The Fmoc deprotection was
performed using a soln. of 20% piperidine in DMF (2 ml/mmol; 3 ꢁ 10 min), with bubbling of N2. After
filtration, the resin was washed with DMF (3 ꢁ 1 min).
Coupling of Amino Acids on Wang Resin (GP 2). The resin was treated with a soln. of Fmoc-
protected amino acid (3 equiv.), HATU (2.9 equiv.) and EtN(i-Pr)2 (5 equiv.) in DMF (3 ml/mmol) for
1 – 4 h under bubbling of N2. After complete coupling (visualized by the TNBS test), the resin was
washed with DMF (5 ꢁ 1 min.).
Wang-Resin Cleavage and Final Deprotection. The dry peptide – resin was taken in a soln. of TFA/
iPr3SiH/H2O (95 :2.5 :2.5, 3 ml/mmol) for 3 h under bubbling of N2. The resin was removed by filtration
and washed with TFA (2 ꢁ 2 ml). The combined filtrate was evaporated under reduced pressure, and the
oily residue was treated with cold Et2O. The precipitated crude peptide was filtered and dried under high
vacuum. Purification by prep. RP-HPLC (2 – 40% in 50 min) and lyophilization yielded peptide 8 (29 mg,
35%) as TFA salt. Colorless solid. Anal. RP-HPLC (2 – 40% A in 35 min, tR 28.1 min, purity > 98%).
1H-NMR (600 MHz, CD3OD): 0.88 – 0.91 (m, 2 ꢁ Me of Ile þ Me of h(F2)hhLeu); 0.97 (d, J ¼ 6.6, Me of
h(F2)hhLeu); 1.12 – 1.19 (m, 1 H, CH2 of Ile); 1.44 – 1.50 (m, 1 H, CH2 of Ile; 1 H, CH2 of Leu); 1.54 – 160
(m, 1 H, CH2 of Leu); 1.60 – 1.65 (m, CH of Leu); 1.78 – 1.85 (m, CH of Ile); 1.91 – 1.97 (m, 1 H, CH2 of
Pro); 1.99 – 2.10 (m, CH2 of Pro, CH2CF2); 2.31 – 2.37 (m, 1 H, CH2 of Pro); 2.41 – 2.49 (m, CH2CO of
h(F2)hhLeu); 2.90 (dd, J ¼ 8.8, 14.1, 1 H, CH2 of Tyr); 3.03 (dd, J ¼ 8.0, 13.6, 1 H, CH2 of Phe); 3.09 –
3.15 (m, 1 H, CH2 of Tyr; 1 H, CH2 of Phe; 1 H, CH2 of His4); 3.28 (dd, J ¼ 7.2, 15.3, 1 H, CH2 of His4);
3.41 (d, J ¼ 5.7, CH2 of His7); 3.54 – 3.58 (m, 1 H, CH2N of Pro); 3.78 – 3.82 (m, 1 H, CH2N of Pro); 4.21
(d, J ¼ 7.7, H ꢀC(a) of Ile); 4.25 – 4.33 (m, CHCF2); 4.56 (dd, J ¼ 6.4, 8.4, HꢀC(a) of Pro); 4.60 – 4.63
(m, HꢀC(a) of Tyr, HꢀC(a) of His7); 4.66 – 4.72 (m, HꢀC(a) of Phe, HꢀC(a) of His4); 6.69 (d, J ¼ 8.7,
2 CH of Tyr); 7.06 (d, J ¼ 8.7, 2 CH of Tyr); 7.20 – 7.24 (m, CH of Phe); 7.26 – 7.31 (m, 4 CH of Phe, CH of
His4); 7.47 (CH, His7); 8.09 (d, J ¼ 8.4, NH of Ile); 8.20 (d, J ¼ 8.2, NH of Tyr); 8.34 (d, J ¼ 9.4, NH of
h(F2)hhLeu); 8.50 (d, J ¼ 7.8, NH of His4); 8.73 (d, J ¼ 1.4, NH of His); 8.75 (m, NH of His); 8.80 (d,
J ¼ 7.0, NH of Phe). 13C-NMR (150 MHz, CD3OD): 11.3 (Me of Ile); 15.8 (Me of Ile); 21.6 (Me of
h(F2)hhLeu); 24.0 (Me of h(F2)hhLeu); 25.4 (CH of h(F2)hhLeu); 25.8 (CH2 of Ile); 26.2 (CH2 of Pro);
26.6 (CH2 of His7); 28.5 (CH2 of His4); 28.7 (CH2CO of h(F2)hhLeu); 30.5 (t, J ¼ 24.3, CH2CF2 of
h(F2)hhLeu); 30.8 (CH2 of Pro); 37.3 (CH2 of Leu); 37.6 (CH2 of Tyr); 37.9 (CH of Ile); 38.4 (CH2 of
Phe); 48.9 (CH2N of Pro); 51.9 (C(a) of His7); 52.3 (t, J ¼ 27.2, CHCF2 of h(F2)hhLeu); 53.8 (C(a) of
His4); 55.2 (C(a) of Tyr); 56.4 (C(a) of Phe); 59.6 (C(a) of Ile); 61.8 (C(a) of Pro); 116.2 (2 ꢁ CH of
Tyr); 118.6 (CH of His4); 120.4 (CH of His7); 128.8 (t, J ¼ 245.4, CF2); 127.9 (C of His7 þ CH of Phe);
129.0 (C of Tyr); 129.5 (2 ꢁ CH of Phe); 130.3 (2 ꢁ CH of Phe); 130.5 (C of His4); 131.3 (2 ꢁ CH of Tyr);
134.9 (CH of His4); 136.2 (CH of His7); 137.9 (C of Phe); 157.2 (COH of Tyr); 167.6 (CO of His7); 172.0
(CO of His4); 173.1 (CO of Phe); 173.7 (CO of Ile); 174.2 (CO of h(F2)hhLeu); 174.8 (2 ꢁ CO, Tyrþ

Helvetica Chimica Acta – Vol. 92 (2009)
1727
Pro). 19F-NMR (375 MHz, CD3OD): ꢀ 109.33 (dm, J ¼ 245.2 Hz, 1 F); ꢀ 111.10 (dm, J ¼ 245.2 Hz,
1 F). HR-MALDI-MS: 1004.5164 ([M þ H]þ, C50H68F2N11Oþ2 ; calc. 1004.51641 (ꢀ1.89 ppm)).
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Received June 5, 2009