The Synthesis of Enantiopure ω‐Methanoprolines and ω‐Methanopipecolic Acids by a Novel Cyclopropanation Reaction: The “Flattering” of Proline

Abstract

COMMUNICATIONS otides in length. In vitro "evolution" was done on this region at 30% mutagenesis. and four more rounds of in vitro selection followed before this second population was cloned. From these sequences, a consensus region was discovered. Certainly though this work is a pioneering achievement in the field, it is an example of how the conventional protocol is significantly more involved than that presented here.In summary, a novel in vitro selection protocol has been designed to take advantage of a combinatorial library of small size that has multiple copies of every distinct sequence. The method condensed the many days of a typical screening strategy to less than two days. This was a proof-of-concept experiment that showed that the new method succeeds by creating a large number of copies of individual sequences in the initial random pool, consistently reducing the level of nonspecific binding sequences per selection round, and effectively amplifying the few surviving sequences.Since only the original synthesized sequences were used for all the screenings, the technique should allow for the iterative in vitro selection of modified oligonucleotides that previously could not undergo this powerful process."01 Hence, this method should significantly increase the power of the in vitro selection method and is the direction that we are currently investigating. Experimental SectionThe DNA library[4] (31 mg) was labeled at the Yend with [y-"P]ATP, purified by gel chromatography, and suspended in 300 mL of folding buffer (300 mM KCI, 5 mM MgCI,. 20mM Tris. pH 7 5 ) . After cooling down to room temperature following denaturation at 75 C, the "P-labeled DNA was loaded onto an acetate-agarose precolumn (300 pL), which was attached directly to a 2.5mM ATP-agarose column (800 pL, Sigma). The precolumn was washed with 600 pL of buffer, and the eluted DNA was allowed to equilibrate on the ATP-agarose column for 10 min. The precolumn was discarded after a single use as were all subsequent columns. After equilibration, the ATP-agarose column was washed with 4 mL of folding buffer to elute unbound or weakly bound oligonucleotides. The retained DNA was eluted with 3 mL of the ATP elution buffer (5mM ATP in folding buffer) and collected in 500 pL fractions.In order to perform another round ofselection, the ATP had to be removed. Hence, the eluted fractions were collected directly into Microcon-3 microcentrifuge devices (3000 D cutoff, Amicon) After membrane diafiltration. about 98% of the total ATP was removed. The filtered fractions were then pooled, and folding buffer was added until a final volume of 10 mL was attained. Theconcentration ofcontaminating ATP concentration was 3 0 p~ for the DNA sample, which was over 80 times more dilute than that of the 2 . 5 m~ ATP-agarose column. Each cycle of selection started with a new set of stacked affinity columns, i.e. a precolumn attached to a ligand column. The screening cycles for the ATP aptamers are summarized in Table 1 The rare-DNA PCR was performed as follows: On the last c...

Catalysis of the Hajos—Parrish—Eder—Sauer—Wiechert Reaction by cis‐ and trans‐4,5‐Methanoprolines: Sensitivity of Proline Catalysis to Pyrrolidine Ring Conformation

“…Theoretical studies of the aldol [6] and the Mannich [7] reactions at UCLA led to the identification of iminium planarity [8] as a key component in proline catalysis. Interestingly, at Montreal the synthesis [9] of 3,4-methanoprolines and their use as conformationally rigid planar proline replacements in enzyme inhibitors has been accomplished. [10] We have now joined forces to explore the use of cis-and trans-4,5-methanoproline as catalysts for the Hajos-ParrishEder-Sauer-Wiechert reaction (Scheme 1) experimentally and computationally.…”

Properties of the Amide Bond Involving Proline 4,5‐methanologues: an Experimental and Theoretical Study

“…Previous studies by our group focused on the synthesis and structural properties of cis ‐ and trans ‐4,5‐methano‐L‐prolines (Figure A) . The inclusion of the methylene bridge to the five‐membered pyrrolidine confers interesting properties to these unnatural proline methanologues.…”

“…[16,[19][20][21] Previous studies by our group focused on the synthesis and structural properties of cis-and trans-4,5-methano-L-prolines (Figure 2 A). [22] The inclusion of the methylene bridge to the five-membered pyrrolidine confers interesting properties to these unnatural proline methanologues. X-Ray structures of both diastereomers show considerable flattening of the pyrrolidine ring, making it essentially planar in the cisdiastereomer, compared to proline itself.…”

Cyclopropane Pipecolic Acids as Templates for Linear and Cyclic Peptidomimetics: Application in the Synthesis of an Arg‐Gly‐Asp (RGD)‐Containing Peptide as an αvβ3 Integrin Ligand

“…So far most cyclopropane amino acids embedded in peptidomimetics have been α‐amino acids,3 in which the three‐membered ring, in addition to locking the χ 1 angle, introduces severe constraints on the proximal backbone torsion angles,4 and β‐amino acids 5. Among α‐amino acids, cyclic compounds like proline3b, 6 and its homologue, pipecolic acid,6a, 7 (Figure 1) have also been fused in a variety of ways to a cyclopropane ring to form bicyclic α‐amino acids,8 with the goal of introducing further constraints in peptidomimetics.…”

“…Compared to proline, the introduction of additional conformational restrictions in this amino acid by merging the piperidine with a cyclopropane ring has concerned very few differently substituted pipecolic acids so far 6a. 7 Extending this approach to (poly)functionalized pipecolic acids would certainly enlarge the scope of these unnatural amino acids in the design and synthesis of highly selective and potent peptide analogues in peptide‐receptor recognition.…”