Synthesis and Conformational Analysis of Fluorine-Containing

Chapter 25

Synthesis and Conformational Analysis of Fluorine-Containing Oligopeptides 1

2

Takashi Yamazaki , Takamasa Kitamoto , and Shunsuke Marubayashi 2

Downloaded by CORNELL UNIV on May 22, 2017 | http://pubs.acs.org Publication Date: January 11, 2007 | doi: 10.1021/bk-2007-0949.ch025

1Strategic Research Initiative

for Future Nano-Science and Technology, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, 2-24-16, Nakamachi, Koganei 184-8588, Japan Department of Applied Chemistry, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Nakamachi, Koganei 184-8588, Japan 2

Preparation of hexapeptide Tyr-D-Ser-Gly-Phe-Leu-Thr possessing an interesting μ-selective opioid activity as well as its F -analog at the Thr terminal methyl group was carried out to investigate the effect of highly electronegative fluorine atoms toward the conformation of the molecules on the basis of information obtained from various types of H NMR techniques. 3

1

Modification of organic molecules with fluorine is one of the major strategies for enhancement or improvement of the original biological activities (1). As already documented (2), this atom is known to be the second smallest and the most electronegative element of all atoms. So, introduction of fluorine atoms into organic molecules imposes only the slightest steric perturbation, but, at the same time, causes possible and significant repulsive or attractive inter action with neighboring electronically negative or positive functional groups, respectively. Such effects including the hydrogen bond (HB) forming ability should result in conformational changes which sometimes lead to a serious decrease in or even loss of the inherent biological activities.

© 2007 American Chemical Society

Soloshonok et al.; Current Fluoroorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

409

410 ,OH

_

H

O

O

IDIH


Tyrosine (Tyr)

A

f

0 H

H

.Ph

H

I

»

0

"

H

D-Serine Glycine Phenyl- • ! _ . Threonine (D-Ser) (Gly) alanine Leucine (Thr) (Phe) (Leu)

Compound la 2a lb 2b

X H H F F

R' H Boc H Boc

J

R H Bn H Bn

Figure 1. Structure of the Target F - and F -Hexapeptides 0

3

Our strong interest in the three dimensional shapes of organic molecules prompted us to initiate a new research project for clarification of the relationship between conformations of molecules and fluorine substitution. For this purpose, the hexapeptide Tyr-D-Ser-Gly-Phe-Leu-Thr l a which was reported to possess interesting ^-selective opioid activity (3) was selected as our target material. The introduction of three fluorine atoms at the methyl moiety of the C-terminal Thr was proposed. Comparison between the F -hexapeptide 2b and native 2a with N- as well as C-protective groups unambiguously demonstrated clear conformational alteration on the basis of extensive H N M R spectroscopy analyses. 3

!

Preparation of the Target Peptides 1 and 2 In Scheme 1 is shown the preparation scheme for Tyr-D-Ser-Gly-Phe-LeuThr l a and its fluorinated analog l b with three fluorine atoms at the Me group of the N-terminal Thr. This peptide formation was mainly carried out by the solution phase method using WSC (4) and HOBt (4) in the presence of triethylamine as the standard condensation conditions. Among various types of condensation required for two amino acids, preparation of dipeptide, Boc-Tyr-DSer-OBn, was problematic and furnished the product only in moderate yield (Entry 1 in Table 1). Change of the solvents with higher polarity (entries 2 and 3), base (entry 4), or the condensation reagent to D P P A (4) (entries 5 to 7)


411

Tyr

Gly

o-Ser

BocBoc-

Boc-

-OBn -OBn

-OH H - -OBn Bocd, 84.6% -OBn Boc-

Boc-

-OH H a 75.7%

-OBn -OBn

t

"OH He. 78.7%

BocDownloaded by CORNELL UNIV on May 22, 2017 | http://pubs.acs.org Publication Date: January 11, 2007 | doi: 10.1021/bk-2007-0949.ch025

-OH H - -OBn a, 97.4% -OBn

•OH Ha, 95.6%

BocBoc-

Leu

Phe Boc-

-OBn -OBn

Boc-

-OH

b, 46.5%

H-

Tyr

Gly

o-Ser

-OH

Phe BocBoc-

BocBocBocBoc-

-OH H d, 84.6%

-OBn Boc-

Leu

-OH He, 95.6%

-OBn Boc-

F -Thr 3

- O H H - -OBn c, 97.4% -OBn -OBn -OBn

88.1% -OBn Boc- - O H

-OH Hc, 94.2%

-OBn Boe-N^oSn

Boc-

-OH H e, 20.0%

Boc-

J

-OBn -OBn

a) WSCHC1, HOBtH 0, Et N/CH Cl , overnight, rt. b)CF C0 H/CH C1 ,1-2h,0°C. c) H , Pd/C/MeOH, 6 h - overnight, rt. d) CDMT, N M M (2 equiv)/AcOEt, overnight, rt. e) WSC-HC1, HOBtH 0, Et N/DMF, overnight, rt. f) (Boc) 0,Na C0 /l,4-dioxane, H 0 , overnight, rt. g) BnBr, NaHC0 /DMF, overnight, rt. 2

3

2

3

2

2

3

2

2

2

2

2

2

2

3

3

Scheme 1. Preparation ofFn- and F Hexapeptides r

la and lb

affected the chemical yields slightly. After some investigation, it was found that C D M T was an appropriate reagent (4) as reported previously by Kaminski (5). This substance worked in a proper manner in the presence of 2.2 equiv of N M M for condensation, D M T - M M (6). N M M played an activating role with (4) where an equimolar amount of base was utilized for the facile conversion of C D M T at ambient temperature to the "true" reagent responsible carboxylic acids and the appropriate amines then attacked to the intermediary ester (Scheme 1). As shown in entry 8, in spite of reaching a similar yield as in entry 1, utilization of AcOEt as the solvent demonstrated significant increase the efficiency of the reaction to attain 84.6% isolated yield in the construction of the desired


412

Table 1. Condensation Conditions of Boc-Tyr and D - S e r - O B n Boc-Tyr-OH + TsOHH-D-Ser-OBn

rt overnight

Condensation Entry Solvent Reagent 1 CH C1 WSCHC1 2 THF WSCHC1 3 DMF WSCHC1 4 DMF WSCHC1 5 DMF DPPA 6 DMF DPPA 7 DMF DPPA 8 THF CDMT 9 AcOEt CDMT 1) For abbreviation names, see ref 4. 0


2

2

MeO

MeO

) r \ N >)=N

N

*

CDMT MeO

3

3

2

3

3

2

c

)~ )=N

MeO

0

3

H

Isolated Yield (%) 42.5 36.6 49.1 27.2 18.0 53.0 58.8 48.5 84.6

(equiv) 1.1 1.1 1.1 1.1 1.3 2.0 2.2 2.2 2.2

0

Base Et N Et N Et N *-Pr NEt Et N Et N /-Pr NEt NMM NMM

3

NMM c l

Boc-Tyr-D-Ser-OBn

14~\ N

0

RC0 H,NMM " -NMMHCI 2

cr DMT-MM

O

V"N >"R N J-0

R*NH

2

*

RC(0)NHR'

MeO Scheme 2. Reaction of CDMT and

DMT-MM

dipeptide Boc-Tyr-D-Ser-OBn with the both hydroxy groups in Tyr and D-Ser unprotected (entry 9). Another advantage worthy o f mention is that this process does not require an inert atmosphere. Trifluorinated Thr was prepared following the procedure of Soloshonok and coworkers (7). JV-benzylated Pro 3 was condensed with o-aminobenzophenone to yield the amide 4 which was then utilized for the formation o f the nickel-Gly complex 5 (Scheme 3). Due to the original stereogenic center in Pro, the Nprotecting benzyl group efficiently blocks the top side of this square-planar N i complex 5, which allowing the diastereoselective si face approach of electrophiles after conversion of 5 to the corresponding enolate. Moreover, in


413

O

J

C F

3 F C


3

1

NHR / 7 (R =R =H) e,f f 68.4% yield 8 (R =R =H) 74.4% yield 1

tAAAAAAAAAAA

6 99.7% yield

V

1

2

2

TS-model

a) o-aminobenzophenone, SOCl /CH Cl , overnight, rt. b) glycine, Ni(N0 ) -6H 0, KOH/MeOH, 1 h, 60 °C. c) CF CHO, MeONa/MeOH, 15 min, 60 °C. d) HCl aqVMeOH, 15 min, reflux, e) B o c A Na C0 /l,4-dioxane, H 0 , overnight, rt. f) BnBr, NaHC0 / DMF, overnight, rt. 2

2

2

3

2

2

3

2

3

2

3

Scheme 3. Preparation of F -Thr 7 3

spite of the sterically more congested environment as opposed to the case when the opposite carbonyl face of C F C H O was used (thus, relationship between H and C F in TS-model is opposite), possible interaction of fluorine with the nickel atom renders the electrophilic trifluoroacetaldehyde accessible to the si face of the carbonyl moiety. Accordingly, the adduct 6 is constructed with the desired correct stereochemistry as the major product. Requisite F -Thr 7 was obtained in 68.4% yield by the simple acidic hydrolysis of the complex 6 and in 55.9% overall yield from the compound 3 in four steps. The diastereoselectivity of F -Thr 7 was determined to be >96.0% de: the F N M R of 7 afforded three sets of doublet peaks due to contamination by a small impurity even after chromatographic purification. We used the integration values of the largest and the second largest peaks to guarantee the least level of selectivity. For the determination of enantio-selectivity, 7 was converted to a diastereomeric mixture of the corresponding a-phenethyl amides giving five peaks by F N M R . This fact led to the conclusion that the selectivity was at least 91.7% ee. 7 was then subjected to the usual Boc protection condition, followed by the base-mediated benzylation of the carboxylic acid (5) to successfully convert it into the Boc-protected benzyl ester 8. 3

3

3

3

19

19


414 To obtain the target hexapeptides l a and l b , one of the most efficient routes would be the combination of the common intermediate TV-terminal pentapeptide and Thr or F -Thr. However, contrary to our expectation, this sequence using both amino acids gave rise to lower chemical yields, 20-40%, along with epimerization of the products which rendered the isolation difficult. After investigation of the order of the condensation while neglecting this efficiency, non-fluorinated 2a was successfully obtained as shown in Scheme 1. The usual deprotection procedure converted 2a into l a in moderate yield. In the case of F Thr after reaction, Boc removal was found to be difficult. Condensation with Boc-Gly-Phe-Leu-OH or even with Boc-Leu-OH disclosed formation of undesirable materials by T L C analysis, apparently proving epimerization possibly at the a-position of F -Thr. A t this position, the proton would be activated by the strongly electron-withdrawing C F moiety. For this reason, along with the fact that we fortunately managed to purify the crude stereoisomeric mixture, condensation of the TV-terminal pentapeptide and F -Thr yielded 2b in 20% isolated yield. The final step was deprotection of the Boc and benzyl groups from 2b, but in spite of our extensive investigation of the reaction conditions, we still did not find out acceptable routes for cleavage at the both terminii. These facts prompted us to compare conformational preference of 2a and 2b by *H N M R . 3

3


3

3

3

Spectroscopic Comparison of 2a and 2b A s described above, with the desired target hexapeptides Boc-Tyr-D-SerGly-Phe-Leu-Thr-OBn 2a and -F -Thr-OBn 2b in hand, one-dimensional *H N M R (9) and then two-dimensional C O S Y and N O E S Y spectra were required for unambiguous assignment of the resonances. In Figure 2 are shown the N O E correlation diagrams for the both materials in DMSO-

Synthesis and Conformational Analysis of Fluorine-Containing

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