Criteria 1.The CF 3 -group has to be attached rigidly to the peptide backbone. 2.The CF 3 -AA should...
1
Criteria 1.The CF 3 -group has to be attached rigidly to the peptide backbone. 2.The CF 3 -AA should be compatible with standard solid phase peptide synthesis (SPPS) protocols, react rapidly and without racemization. 3. To replace a natural amino acid, the CF 3 -AA must not perturb the characteristics of the peptide (polarity, conformation, function). our ``new`` 19 F-NMR labels CF 3 NH 2 COOH N COOMe O O CF 3 CHN 2 N COOMe O O CF 3 H H H N COOMe O O CF 3 H H H N COOMe O O H H H CF 3 N H CF 3 H H COOH H NH 2 + CF 3 H H H COOH NH 2 + H H H CF 3 COOH CF 3 C O O- CF 3 C O O- N H CF 3 H H COOH N H CF 3 H H COOH N COOMe O O CF 3 CHN 2 , CuO Tf N COOMe O O CF 3 H H CF 3 N COOMe O O CF 3 H H CF 3 N COOMe O O CF 3 CF 3 CHN 2 quant. quant. C uC l + + 1)N aO H ,M eO H 2)TFA,D C M 3)D ouex-50 1)N aO H ,M eO H 2)TFA,D C M 1)N aO H ,M eO H 2)TFA,D C M 1)H Brreflux 2)D ouex-50 + 1)H Brreflux 2)D ouex-50 C uC l hexane 70% 90% 30% 50% 50% Synthesis of novel CF 3 -substituted α-amino acids as 19 F-NMR labels P. K. Mykhailiuk a , S. Afonin b , N. Gvozdovska a , P. Wadhwani b , M. Berditsch c , M. Ieronimo c , S. L. Grage b , A. S. Ulrich b,c , I. V. Komarov a a Kyiv National Taras Shevchenko University, Kyiv, Ukraine b Institute of Biological Interfaces, Forschungszentrum Karlsruhe, Karlsruhe, Germany c Institute of Organic Chemistry, University of Karlsruhe, Karlsruhe, Germany Criterion 1: rigid structure (X-ray analysis) CF 3 N H OH Ph CN N COOMe O O CF 3 H H H Introduction CF 3 -substituted α-amino acids (CF 3 -AA) are highly useful as 19 F-NMR labels. They are especially informative in solid-state NMR studies of peptides in biomembranes. To be a ``good`` label the CF 3 -AA should meet several criteria. NH 2 COOH CF 3 CF 3 NH 2 COOH NH 2 COOH F 3 C C H 3 previously used 19 F-NMR labels CF 3 -Bpg CF 3 -Phg Criterion 3: unperturbed peptide (CD, biological function) 9-(CF 3 Bpg)-PGLa A single CF 3 -Bpg label was substituted in four different positions of the α-helical peptide PGLa: GMASKAGAI 9 A 10 GKI 13 A 14 KVALKAL- NH 2 Circular dichroism showed that a single CF 3 -Bpg label does not perturb the conformation of the peptide. 14-(CF 3 Bpg)-PGLa 10-(CF 3 Bpg)-PGLa 13-(CF 3 Bpg)-PGLa wild type-PGLa The antimicrobial α-helical peptide PGLa was used to test the incorporation of the „old“ CF 3 -Phg and the „new“ CF 3 -Bpg label. The antimicrobial function of the four labelled analogues of PGLa was tested, and all minimal inhibitory concentrations were found to be identical to the wild type peptide Cl Cl Cl Cl Br Br CF 3 O OH CF 3 I CF 3 N H OH Ph CN CF 3 O H CF 3 N H OH Ph CN C H Br 3 M eLi CF 3 I PbAc 4 CF 3 NH 2 COOH PbAc 4 CF 3 NH 2 COOH + N aO H 1)t-BuLi 2)HCO O M e R-phenylglycinol TMSCN + HCl -78 C M eO H 1) 2) HCl 1) 2) 65% (2 steps) 85% (2 steps) 95% 95% Synthetic schemes References 3. Mykhailiuk, P. K. et al, Angew. Chem. Int. Ed. 2006, 45, 5659-5661. 4. Afonin, S. et al, J. Pept. Sci. 2007, in print. 5. Mykhailiuk, P. K. et al, Angew. Chem. Int. Ed. 2007, submitted. 1. Ulrich A. et al, Prog. NMR Spectr. 2005, 46, 1-21. 2. Glaser R. et al, Biophysical Journal 2005, 88, 3392-3397. Contact: [email protected] Several novel conformationally restricted CF 3 -labelled α-amino acids have been synthesized. These amino acids were designed as 19 F-NMR labels for solid state NMR studies of membrane-bound peptides. CF 3 -Bpg has been demonstrated to fulfil all criteria for optimal use as a 19 F-NMR. The application of the new CF 3 - Pro analogues as 19 F-NMR labels is currently under investigation. Conclusions F 3 -Aib F 3 -Ala CF 3 (3/4)- Pro CF 3 (4/5)- Pro Criterion 2: successful peptide synthesis (RP-HPLC) HPLC of 13-(CF 3 -Phg)-PGLa L-(CF 3 -Phg) racemizes during peptide synthesis. 2 diastereomers of 13- (CF 3 -Phg)-PGLa (containing D- and L-CF 3 -Phg) are formed. HPLC of 13-(CF 3 -Bpg)-PGLa L-(CF 3 -Bpg) does not racemize during solid phase Fmoc peptide synthesis. RT (min) RT (min) λ (nm) MRE -15 -10 -5 0 5 10 15 20 25 30 180 190 200 210 220 230 240 250 260 PGLa-wt 15 16 17 18
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Transcript of Criteria 1.The CF 3 -group has to be attached rigidly to the peptide backbone. 2.The CF 3 -AA should...
Criteria 1. The CF3-group has to be attached rigidly to the peptide backbone. 2. The CF3-AA should be compatible with standard solid phase peptide
synthesis (SPPS) protocols, react rapidly and without racemization.3. To replace a natural amino acid, the CF3-AA must not perturb the
characteristics of the peptide (polarity, conformation, function).
our ``new`` 19F-NMR labels
CF3
NH2
COOH
NCOOMe
OO
CF3CHN2
NCOOMe
OO
CF3
H
H
H
NCOOMe
OO
CF3
H
H
HN
COOMe
OO
H
H
HCF3
NH
CF3
H
HCOOH
H
NH2+CF3
H
H
H
COOHNH2+
H
H
HCF3
COOH
CF3COO- CF3COO-
NH
CF3
HH
COOHNH
CF3
HH
COOH
NCOOMe
OO
CF3CHN2, CuOTf
NCOOMe
OO
CF3
HH
CF3
NCOOMe
OO
CF3
HH
CF3
NCOOMe
OOCF3
CF3CHN2
quant. quant.
CuCl
+ +
1) NaOH, MeOH2)TFA, DCM3) Douex-50
1) NaOH, MeOH2)TFA, DCM
1) NaOH, MeOH2)TFA, DCM
1) HBr reflux2) Douex-50
+
1) HBr reflux2) Douex-50 CuCl
hexane
70%
90%
30%
50% 50%
Synthesis of novel CF3-substituted α-amino acids as 19F-NMR labels
P. K. Mykhailiuka, S. Afoninb, N. Gvozdovskaa, P. Wadhwanib, M. Berditschc, M. Ieronimoc, S. L. Grageb, A. S. Ulrichb,c, I. V. Komarova
aKyiv National Taras Shevchenko University, Kyiv, UkrainebInstitute of Biological Interfaces, Forschungszentrum Karlsruhe, Karlsruhe, GermanycInstitute of Organic Chemistry, University of Karlsruhe, Karlsruhe, Germany
Criterion 1: rigid structure(X-ray analysis)
CF3NH OH
Ph
CN
NCOOMe
OO
CF3
H
H
H
IntroductionCF3-substituted α-amino acids (CF3-AA) are highly useful as 19F-NMR labels. They are especially informative in solid-state NMR studies of peptides in biomembranes. To be a ``good`` label the CF3-AA should meet several criteria.
NH2
COOHCF3
CF3NH2
COOH
NH2
COOH
F3C
CH3
previously used 19F-NMR labels
CF3-BpgCF3-Phg
Criterion 3: unperturbed peptide(CD, biological function)
9-(CF3Bpg)-PGLa
A single CF3-Bpg label was substituted in four different positions of the α-helical peptide PGLa: GMASKAGAI9A10GKI13A14KVALKAL-NH2
Circular dichroism showed that a single CF3-Bpg label does not perturb the conformation of the peptide.
14-(CF3Bpg)-PGLa
10-(CF3Bpg)-PGLa
13-(CF3Bpg)-PGLa
wild type-PGLa
The antimicrobial α-helical peptide PGLa was used to test the incorporation of the „old“ CF3-Phg and the „new“ CF3-Bpg label.
The antimicrobial function of the four labelled analogues of PGLa was tested, and all minimal inhibitory concentrations were found to be identical to the wild type peptide
Cl
Cl
Cl
Cl
BrBr
CF3
O
OH
CF3 I
CF3
NH OH
Ph
CN
CF3
O
H
CF3
NH OH
Ph
CN
CHBr3
MeLi CF3I
PbAc4
CF3
NH2
COOH
PbAc4
CF3
NH2
COOH
+
NaOH
1)t-BuLi2)HCOOMe
R-phenylglycinol
TMSCN
+
HCl
-78 C
MeOH
1)
2)HCl
1)
2)
65%(2 steps)
85%(2 steps)
95%95%
Synthetic schemes
References
3. Mykhailiuk, P. K. et al, Angew. Chem. Int. Ed. 2006, 45, 5659-5661.4. Afonin, S. et al, J. Pept. Sci. 2007, in print.5. Mykhailiuk, P. K. et al, Angew. Chem. Int. Ed. 2007, submitted.
1. Ulrich A. et al, Prog. NMR Spectr. 2005, 46, 1-21.2. Glaser R. et al, Biophysical Journal 2005, 88, 3392-3397.
Contact: [email protected]
Several novel conformationally restricted CF3-labelled α-amino acids have been synthesized. These amino acids were designed as 19F-NMR labels for solid state NMR studies of membrane-bound peptides. CF3-Bpg has been demonstrated to fulfil all criteria for optimal use as a 19F-NMR. The application of the new CF3-Pro analogues as 19F-NMR labels is currently under investigation.
Conclusions
F3-Aib
F3-Ala
CF3(3/4)-Pro
CF3(4/5)-Pro
Criterion 2: successful peptide synthesis(RP-HPLC)
HPLC of 13-(CF3-Phg)-PGLa L-(CF3-Phg) racemizes during peptide synthesis. 2 diastereomers of 13-(CF3-Phg)-PGLa (containing D- and L-CF3-Phg) are formed.
HPLC of 13-(CF3-Bpg)-PGLaL-(CF3-Bpg) does not racemize during solid phase Fmoc peptide synthesis.
RT (min)
RT (min)
λ (nm)
MR
E
-15
-10
-5
0
5
10
15
20
25
30
180 190 200 210 220 230 240 250 260
PGLa-wt
15
1617
18
