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21.11.6.2.2 Variation 2: Auxiliary-Mediated Chemical Ligation

DOI: 10.1055/sos-SD-021-00693

Lubell, W. D.; Blankenship, J. W.; Fridkin, G.; Kaul, R.Science of Synthesis, (200521792.

To extend the utility of chemoselective ligation for making amide bonds, research has focused on auxiliary-mediated native chemical ligation,[‌307‌,‌499‌‌503‌] aiming to remove the ­requirement of an N-terminal cysteine residue by moving the thiol functionality to an N-linked auxiliary. Pioneering research using the thiol capture strategy had shown that auxiliary-mediated ligation was possible using a C-linked 4,6-disubstituted dibenzofuran[‌504‌‌506‌] and a peptide with an N-terminal cysteine residue. The N-linked auxiliary approach mimics native chemical ligation: the thioester exchanges onto the thiol of the auxiliary, then migrates from the sulfur to the nitrogen of the amine terminus. However, unlike native chemical ligation, the auxiliary must then be removed by selective deprotection to generate the native amide bond. Rates of acyl transfer are usually contingent on ring size of the auxiliary; most auxiliaries transfer the acylating fragment via a six-membered transition state, which usually proceeds at a relatively slower rate of acyl transfer than the five-membered counterpart in native chemical ligation. Additionally, acylating a secondary amine is generally more difficult than a primary amine. Success using this strategy has necessitated strict requirements on the size of the auxiliary and the use of preactivated thioesters. Two general classes of auxiliaries have been developed. The first class developed from the 2-sulfanylbenzyl auxiliary 1,[‌502‌] and includes the 4,5-dimethoxy-2-sulfanylbenzyl 2 [Dmb(2-SH)] and 4,5,6-trimethoxy-2-sulfanylbenzyl 3 [Tmb(2-SH)] auxiliaries (Scheme 42),[‌499‌,‌500‌,‌507‌] which offer reasonable reaction rates, and have extended the range of potential ligation junctions to Xaa-Gly and Gly-Xaa (where Xaa is a proteinogenic amino acid other than proline). Dmb(2-SH) 2 can be removed by treatment with anhydrous hydrogen fluoride after ligation, and Tmb(2-SH) 3 can be cleaved by trifluoroacetic acid after ligation.[‌499‌] The second class of auxiliaries, developed from the 1-amino-1-phenyl-2-sulfanylethyl group 4,[‌501‌] offer similar reactivity as the first class; moreover, analogues have been developed that can undergo photochemical deprotection (e.g., 5[‌501‌,‌508‌,‌509‌] and 6[‌509‌]) or deprotection by trifluoroacetic acid (e.g., 7).[‌307‌,‌510‌] One limitation of this class of racemic auxiliaries (47) are the lengthy syntheses, which produce mixtures of enantiomers such that peptide coupling produces diastereomeric products, which may complicate purification and analysis. The configuration of the auxiliary, however, has been reported to cause no effect on the rate of ligation.[‌509‌] Preactivation of the thioester is necessary for ligation of sterically demanding residues (other than Gly-Gly, His-Gly, Cys/nbhy;Gly) using these auxiliaries, because free thiols can inhibit ligation;[‌499‌] for less ­sterically demanding ligations, TAMPAL thioesters have been used successfully. The syntheses of N-(9-fluorenylmethoxycarbonyl)-N-{2,3,4-trimethoxy-6-[(4-methoxybenzyl)sul­fanyl]benzyl}glycine[‌509‌] and 1-(4,5-dimethoxy-2-nitrophenyl)-2-[(4-methylbenzyl)sulfan­yl]ethylamine[‌509‌] are shown in Schemes 43 and 44, respectively.

Meeeee 88 Meeeeeeee eee Meeeeeeee-Meeeeeee Meeeeeee Meeeeeee[‌888‌,‌888‌‌888‌,‌888‌‌888‌]

Meeeee 88 Meeeeeeee ee M-(8-Meeeeeeeeeeeeeeeeeeeeeee)-M-{8,8,8-eeeeeeeeee-8-[(8-eeeeeeeeeeeee)eeeeeeee]eeeeee}eeeeeee[‌888‌]

Meeeee 88 Meeeeeeee ee 8-(8,8-Meeeeeeee-8-eeeeeeeeeee)-8-[(8-eeeeeeeeeeee)eeeeeeee]eeeeeeeeee[‌888‌]

Meeeeeeeeeee Meeeeeeee

Meeeeee α-Meeeee Meeeeeeeee; Meeeeee Meeeeeeee:[‌8‌,‌888‌,‌888‌]

Meeeeeee eeeeeeee eeee eeeeeeeeeee ee eee eeeeeeeeeee Mee-eeeeeeeee-M-eeeeee.[‌888‌] Meeeeeeeeeeee, eeeeeeeee eeeeeeee eeee eeeeeeeeeee ee MMMMMM eeeee[‌888‌] (eee eeee Meeeeee 88.88.8.8.8) eee eeeeeeeee eeee eeeeeeeee ee eeeeeeeeee eee eeeee eeeeeee ee 8M eeeeeeeee eeeeeeeeeeeee eeeeeeeeee 8.8M Me8MM8 (eM 8.8) eee 8.8M (MM8)8M. [(MM8)8M eee eeeeeeeeee eeee eee eeeeeeeee eeeeee ee eeeeeeee ee 8.8M (MM8)8M; eee eeee eee eeeeeeee ee eee eeeee eM eeeee eeee eeeeeeee.] Meeeeeeeee eee eeeeeeeee ee eeeeeeeeee MMMM. Meeeeee α-eeeeee eeeeeeeeee eeee eeeeeeee eeeee eeeeeeeeee eee eeeeeee eeeeeeee (888ee, eeeeeeeee 8.88eeee) eee 8,8-eeeeeeeee(8-eeeeeeeeeeee eeee) (88 eeeee, eeeeeeeee 88ee, 8.8eeee) ee e eeee ee 8M eeeeeeeee eeeeeeeeeeeee (88eM) eeeeeeeeee 8.8M Me8MMM8 ee eM 8.8. Mee eeeeeee eee eeeeeeee eeee e eeeeee eeeeee eeeeeee eee eee eeeee eee 88eee eeeeee eeeeeeeee eeee 8% MeMM. Mee eeeeeeee eeeeeeee eee eeeeeeeee ee eeeeeeeeee MMMM eee eeeeeeee eeeee eeeeeeeeeeeeeee MMMM. Meeee eeeeeeeeeeeeee, eee eeeeeee α-eeeeee eeeeeeeeee eeee eeee eeeeeeeeeee ee eeeeee eeeee eeeeeee eeeeeeee; eeeee: eee eeeeeeee.

M-(8,8,8-Meeeeeeeee-8-eeeeeeeeeeeeee)eeeeeee eee Meeeeeeeeeeee eeee Meeeeeee:[‌888‌]

8,8,8-Meeeeeeeeeeeeeee Meeeeeeeeeeeeeeeeeeeeeeee (8):

8,8,8-Meeeeeeeeeeeeeee (8e, 88eeee) eee eeeeeeeee ee eeeeeee (88eM) eee 88% M8MM8 eeee (88eM) eee eeeee. Mee eeeeeee eee eeeeee ee 8°M eee MMMM (8.8eM, 88eeee) eee eeeee eeeeee eeeeeeee eeee eeeeeeee ee eeeeeeee eee eeeeeeeeeee eeeee 88°M. Mee eeeeeee eee eeeeeee ee eeee ee ee eee eeeeeee eeeeeee eee 8e, eeee eeeeeeeee eeee eeeeeee (8 × 88eM). Mee eeeeeeee eeeeeee eeeeeeee eeee eeeeee eeee M8M (88eM), eeeee (MeMM8), eee eeeeeeeeeeee ee eeee eee eeeeeeeeeeeeeeeeeeeeeeeee ee e eeee eeeeee eeeee eeeee eee eeee eeeeeee eeeeeee eeeeeeeeeeee; eeeee: 8.88e (88%); 88M MMM (888.8MMe, MMMe8, δ): 88.8.

8,8,8-Meeeeeeeee-8-[(8-eeeeeeeeeeeee)eeeeeeee]eeeeeee (8):

Meeeeeeeeeeeeeeeeeeeeeeee 8 (888ee, 8.88eeee) eee eeeeee ee e eeeeeeeee eeee eee 8eee% Me(MMe)8 eee 8eee% (+)-(M)-MMMMM eeee eeeee. Mee eeee eee eeeeee eeee e eeeee eee eeee eeeeee, eeee MMM (8eM), Mee-MM (88 µM, 8.88eeee), eee Me8M (88 µM, 8.88eeee) eeee eeeee eeeee eeeee. Mee eeee eee eeeeee ee e MMM Meeeeeee eeeeeeeee eeeeeeee eeee MeeeMeeeee eeeeeeee eee eeeeee ee 888°M ee e eeeeeeeee eeeee ee 888M eee 88eee. Mee eeeeeee eee eeeeeee eeee MeMMe (88eM) eee eeeeee eeee eeeee (88eM), eeee eeeee (MeMM8), eeeeeeee, eee eee eeeeeee eeeeeee eeeee eeeeeee eeeeeeee. Mee eeeee eeeeeee eee eeeeeeee ee eeeeee eeeeeeeeeeeeee [eeeeee eee, MeMMe/eeeeeeeee eeeee (ee 8888°M) 8:8] ee eeee eee eeeeeee ee e eeeeee eeeee eeeee; eeeee: 88ee (88%).

8,8,8-Meeeeeeeee-8-[(8-eeeeeeeeeeeee)eeeeeeee]eeeeeeeeeeee (88):

Meeeeee 8 (888ee, 8.88eeee) eee eeeeeeeee ee eeeee MM8Me8 (8.8eM) eee eeeee ee e eeeee-eeeee eeeee eeeeee eeee e eeeeeeeee eeeee eeeee, eeeeeee eeee eeeee MMM (8.88eM, 8.8eeee), eeeeee ee 8°M, eee eeeeeee eeeeeeee eeee MMMe8 (8.88eM, 8.8eeee) eeee 88eee. Mee eeeeeeee eee eeeeeee ee eeee ee ee, eeeeee ee 888°M eee 8e, eeeeeee ee eeee, eeeeeee eeeeeeee eeee eee. MeMMM8 (88eM), eee eeeeeee ee ee eee 8e. Mee eeeeeee eee eeee eeeeeeeee eeee MM8Me8 (8 × 88eM). Mee eeeeeeee eeeeeee eeeeeeee eeee eeeeee eeee eeeee (88eM), eeeee (MeMM8), eee eeeeeeeeeeee eeeee eeeeeee eeeeeeee ee eeee e eeeee eeeee, eeeee eee eeeeeeee ee eeeeee eeeeeeeeeeeeee [eeeeee eee, MeMMe/eeeeeeeee eeeee (ee 8888°M) 8:8] ee eeee eee eeeeeee ee e eeeee eeeee; eeeee: 888ee (88%).

eeee-Meeee M-{8,8,8-Meeeeeeeee-8-[(8-eeeeeeeeeeeee)eeeeeeee]eeeeee}eeeeeeeee (88) ee Meeeeeeee Meeeeeeee eeee ee Meeee Meee:

Mee eeeeeeee 88 (888ee, 8.88eeee) eee eeeeeeeee ee MM8Me8 (88eM) eee 8% MeMM (8.8eM). Mee eeeee eeee eeee-eeeee eeeee (8.88eeee) eee eeeee, eeeeeeee ee MeMM(MMe)8 (888ee, 8.88eeee). Mee eeeeeee eee eeeeeee ee ee eee 8e, eeee eeeeeeeeeee eeee eee. MeMMM8 (88eM). Mee eeeeeee eee eeeeeeeee eeee MM8Me8 (8 × 88eM), eeeeee eeee eeeee (88eM), eeee eeeee (MeMM8), eeeeeeee, eee eeeeeeeeeeee ee e eeeeee eee. Mee eeeee eeeeeeee eeee eeeeeeee ee eeeeee eeeeeeeeeeeeee [eeeeee eee, MeMMe/eeeeeeeee eeeee (ee 8888°M) 8:88] ee eeeeee eee eeee eeeeeee, eeeeeeeee ee e eeeee eeee; eeeee: 88%.

M-(8-Meeeeeeeeeeeeeeeeeeeeeee)-M-{8,8,8-eeeeeeeeee-8-[(8-eeeeeeeeeeeee)eeeeeeee]eeeeee}eeeeeee (88):

Mee eeeee 88 eee eeeeeee eeee MMM/M8M (88:8) ee 8°M. Mee eeeeeee eee eeeeeee ee 8°M eee 8e, eeeeeeeeeeee eeeee eeeeeee eeeeeeee, eee eeeeeeeeee eeee eeeeeee. Mee eeeee M-{8,8,8-eeeeeeeeee-8-[(8-eeeeeeeeeeeee)eeeeeeee]eeeeee}eeeeeee eee eeee eeeeeeee ee eee eeee eeee.

Mee eeeeeeeeeeeee (888ee, 8.88eeee [eeeeeee]) eee eeeeeeeee ee M8M (8eM), eeeeeee eeee Me8M (88 µM, 8.88eeee), eeeeeeee ee e eeee ee Meee-MMe (88ee, 8.88eeee) ee MeMM (8eM) eee eeeeeee ee ee eee 8e, eeeeeeeeeee e eM ee 8.88.8 ee eee eeeeeeee ee eeee Me8M ee eeeeeeeee. Mee eeeeeee eee eeeeeeeeeee eeee 8.8M MMe eee eeeeeeeee eeee MM8Me8 (8 × 88eM). Mee eeeeeeee eeeeeee eeeeeeee eeee eeeeee eeee 8.8M MMe (88eM) eee eeeee (88eM), eeeee (MeMM8), eeeeeeee, eee eeeeeeeeeeee eeeee eeeeeee eeeeeeee ee eeee e eeeeee eee, eeeee eee eeeeeeee ee eeeeee eeeeeeeeeeeeee [eeeeee eee, MeMMe/eeeeeeeee eeeee (ee 8888°M) 8:8] ee eeee e eeeee eeee ee eee eeeeeee; eeeee: 888ee (88% eeee 8 eeeee).

M-Mee Meeeeeeeeeee ee M-Meeeeeee M-{8,8,8-Meeeeeeeee-8-[(8-eeeeeeeeeeeee)eeeeeeee]eeeeee}eeeeeee eeeee Meeeeee Meeeeeeee eee Meeeeeeeeeee:

Mee M-Mee-eeeeeeeee eeeeeee (8.8ee) eee eeeeeeeee ee 88% MeMM (8.8eM) ee 8°M, eeeeeee eeee Me(MMe)8 (8.8ee), eeeeeeee eeeeeee eeeeeeeeee ee eeeee eeeeeeeeeeeeee ee e eeeee eeeeeeeeeeeee ee 8% e/e. Mee eeeeeee eee eeeeee (888 eee) ee ee eeeee eeeee. Meeee 8e, MM-MM eeeeeeee eeeeeeeee eeee eee eeeeeeee eee eeeeeeee ee ee eeeeeeee eeeeeeee eee eeeeeeee. Mee eeeee eeeee eeeeeeeeeee eee eeeeeee ee eeeeeeeeeeeeee ee 88,888 eee eee 8eee. Mee eeeeeeeee eeee eee eeeeeeee eeeeeeee ee eeeeeeeeeeeeeee MMMM eee eee eeeeeeeee eeeeeeeee eeee eeeeeeeeeee ee eeeeee eee eeeee ee e eeeeee eeeee eeeee; eeeee: eee eeeeeeee.

8,8,8-Meeeeeeeee-8-eeeeeeeeeeeeee-Meeeeeee Meeeeeee Meeeeeee eee Meeeeeeeeeee; Meeeeee Meeeeeeee:[‌888‌]

Meeeeeee: Mee eeeeeeeee-eeeeee eeeeeee eee eeeeeeeee ee eeeeeeee eeeeee [8M eeeeeeeee eeeeeeeeeeeee/888 eM Me8MM8, eM 8.8/8% eeeeee 8-eeeeeeeeeeeeeeeeeeeeeee/88 eM eeee(8-eeeeeeeeeeee)eeeeeeeee] (8.888.8eM) ee e eeeee eeeeeeeeeeeee ee 8 eM (eeeeeeeee 8ee·eM8). Meee eeee eee eeee eeeeeeeeeee ee ee Meeeeeeee eeee eeeeeeeeee eee eeeeeeeeeee eeeeeee eeeeeeeee (8:8 eeeee ee eeeeeeeee eeeeeeeee). Mee eeeeeee eee eeee eeeeee ee ee (888 eee) eeeee eeeee eee eee eeeeeeee eee eeeeeeeee eeeee MM-MM. Meeeeeee eeeeeeee eeee eeeeeeee eeeee MMMM, eee eeeeeeeeeee ee eeee eee eeeeeeee; eeeee: eee eeeeeeee.

Meeeeeeee eeeeeee: Mee eeeeeeee eeeeeeee eeeeeeee eeee eeeeeeeee ee 88% MMM eee eeeeeee ee eeeee eeee eeeeeeeeee eeeeeeeee ee ee eee 8e. Meeee eeee eeee eee eeee ee eee MMM eee eeeeeee eeeee e eeeeee ee eeeee (eeee eeee eeeeeeee eeeeeee eee eeeeeeeee eeeeeee eeeeeeee) eee eee eeeeeee eeeeeee eee eeeeeeeeeeee eeee eee-eeee Me8M. Mee eeeeeeeeeeee eeeeeee eee eeeeeeeee ee eeeeeeeeeeeeee eee eeeeeeee ee eeeeeeeeeeeeeee MMMM; eeeee: eee eeeeeeee.

8-(8,8-Meeeeeeee-8-eeeeeeeeeee)eeeeeeeeeee Meeeeeeee eee Meeeeeeeeeeee eeee Meeeeeee:[‌888‌]

8-Meeee-8,8-eeeeeeeeeeeeeeee (88):

Me e eeeeeeeeee ee Me8M+MeMe (88.8e, 88.8eeee) ee MMM (888eM), 8M MeMMMM ee MMM (88eM, 88eeee) eee eeeee eeeeeeee eeee 88eee ee 8°M eee eeeeeee eee 8e. M eeee ee 8,8-eeeeeeeee-8-eeeeeeeeeeeeeeeee (88; 88e, 88.8eeee) ee MMM (888eM) eee eeee eeeee eeeeeeee ee eee eeeeeee, eeeee eee eeee eeeeeee ee ee eee ee eeeeeeeeee 88e eee eeeeeeeeeeee. Mee eeeeeee eee eeeeeeeeeee eeeeeee MMMe8 (888eM) eee eee. ee MM8Me (888eM). Mee eeeeee eeee eeeeeeeee eee eee eeeeeee eeeee eee eeeeee eeee eee. ee MM8Me (8 × 888eM) eee eeeee (8 × 888eM), eeeee (Me8MM8), eee eeeeeeeeeeee eeeee eeeeeee eeeeeeee. Meeeee eeeeeeeeeeeeee ee eee eeeeeee (eeeeee eee, MeMMe/eeeeeeeee eeeee 8:8) eeeeeeee eee eeeeeee; eeeee: 8.8e (88%); Me 8.88 (eeeeee eee, MeMMe/eeeeeeeee eeeee 8:8).

8-(eeee-Meeeeeeeeeeeeeeeeee)-8-(8,8-eeeeeeeee-8-eeeeeeeeeee)eeeeeee (88):

Me e eeeeeee eeee ee eeee-eeeee eeeeeeeee (888ee, 8.8eeee) ee MeMM (88eM), 8.8M ee MeMM (8.8eM) eee eeeeeee eeeeeeee e-MeMMe[‌888‌] (8.8eM) eeee eeeeeeeeeeee eeeee eee eee eeeeeeeee eeeeeee eee eeeeeee eee 8eee. Mee eeeee eee eeeeee eeee ee eee eeeeee eee eee eeeeeee eee eeeeeee eeeeeeeeeeee eeee (MMM)8MMMM (eeeeeeeeeeee eeeeeeeeeee-8,8-eeee eeeeeee; 888ee, 8.88eeee) ee MeMM (88eM), eeeeeee 88 (888ee, 8.8eeee) ee MeMM (88eM), eee M8MeM8(MM)8 (88ee, 8.8eeee), eeeeeee ee 8°M eee 88e, eee eeeeeeee eeee eee. ee Me8M (88eM). Mee eeeeee eeee eeeeeeeee eee eee eeeeeee eeeee eee eeeeeeeee eeee MeMMe (8 × 888eM). Mee eeeeeeee eeeeeee eeeeeeee eeee eeeeee eeee eeeee (8 × 888eM), eeeee (Me8MM8), eee eeeeeeeeeeee. Meeeee eeeeeeeeeeeeee ee eee eeeeeee (eeeeee eee, MeMMe/eeeeeeeee eeeee 8:8) eeeeeeee eee eeeeeee; eeeee: 888ee (88%); Me 8.88 (MeMMe/eeeeeeeee eeeee 8:8); ee eeee ee eee eeeeeeeee eeeeeee 88 (888ee, 88%).

M-[8-(eeee-Meeeeeeeeeeeeeeeeee)-8-(8,8-eeeeeeeee-8-eeeeeeeeeee)eeeee] Meeeeeeeeeeee (88):

Me ee eee-eeee, eeeeeee eeee ee Me8M (888ee, 8.8eeee) ee MMM (88eM), eeeeeeeeeee eeeeeeeeeeeeeeee (MMMM; 8.88eM, 8.8eeee) eee eeeee. Mee eeeeeee eee eeeeeee eee 88eee, eeeeee eeeee eeee e eeeee eeeeeeeeeee eee eeeeee. M eeee ee eeeeeee 88 (888ee, 8.8eeee) ee MMM (88eM) eee eeeeeeeeee M-eeee (8.88eM, 8.8eeee) eeee eeeeeeeeeeee eeeee eeeeeeee eee eee eeeeeee eee eeeeeee ee ee eee ee eeeeeeeeee 88e. Mee eeeeeee eee eeeeeee eee eee eeeeeee eee eeeeeeeee ee eeeeeee (888eM) eee eeee eeeeeeee ee 8°M eee 88e. Mee eeeeeeeeeee eee eeeeeeee eee eee eeeeeee eee eeeeeee. Meeeee eeeeeeeeeeeeee ee eee eeeeeee (eeeeee eee, MeMMe/eeeeeeeee eeeee 8:8) eeeeeeee eee eeeeeee; eeeee: 888ee (88%); Me 8.88 (MeMMe/eeeeeeeee eeeee 8:8).

M-(eeee-Meeeeeeeeeeeee)-8-(8,8-eeeeeeeee-8-eeeeeeeeeee)-8-[(8-eeeeeeeeeeee)eeeeeeee]eee­eeeeeee [88, M8=Mee(8-Me)]:

Me e eeeeeee eeee ee 88 ee eee MeMM (888eM), 8M MeMMe ee MeMM (8.88eM, 8.8eeee) eee eeeee eee eee eeeeeeeee eeeeeee eee eeeeeee eee 8eee. Mee(8-Me)Me (888ee, 8.8eeee) eee eeeee eee eee eeeeeee eee eeeeeee eee ee eeeeeeeeee 88eee. Mee eM eee eeeeeeee ee 8 ee eeeeee Meeee M+. Mee eeeee eee eeeeeeee eee eee eeeeeee eee eeeeeee. Meeee eeeeee eeeeeeeeeeeeee ee eee eeeeeee (eeeeee eee, MeMMe/eeeeeeeee eeeee 8:88) eeeeeeee eee eeeeeee; eeeee: 888ee (88%); Me 8.8 (MeMMe/eeeeeeeee eeeee 8:8).

8-(8,8-Meeeeeeee-8-eeeeeeeeeee)-8-[(8-eeeeeeeeeeee)eeeeeeee]eeeeeeeeee (88):

Mee-eeeee 88 eee eeeeeeeee ee eeee MMM, eeeeeee eee 8eee, eee eee eeeeeee eee eeeeeee eeeee eeeeeee eeeeeeee; eeeee: eee eeeeeeee. Mee eeeee eeeeeee eee eeee eeeeeee eeeeeee eeeeeeeeeeee ee eee eeeeeeeee eeeeeeee eeee eee eeeeeeeeeee eeeeeee.

Meeeeeeeeeeeeeee ee Meeeeee Meeee; Meeeeeee ee Meeeeeeee ee Meeeeee Meeee:

MMMMMMM: Meeeeeeeeeeee eee eeeeee eee, eeee, eee eeeeeeeeeee eeeee eeeeeeeee, eee eeee eeeeeeeeeee. Meee eeeeee eeee ee eeeeeee eeee eeeeee ee e eeeeeeee.

Meeee eeeeeee ee eee eeeeeeeeeee Mα-Mee ee Meee eeeee, eee eeeeeee eeeee eee eeeeeeeeeee eeee eMe8MMe eee eeeeee eeee MMM eee MM8Me8. M eeee ee eee α-eeeee eeeeeeeee eeeeeeeee ee MM8Me8 (88 eeeee eeeeeeee ee eeeee eeeeeee) eee eeeeeeee ee eeeeeeee e 8°M eeee ee eeeeeeeeeee eeee ee MM8Me8 eeee MMM (88 eeeee), eee eee/eeee eeee eee eeeeeee, eee eee eeeeeee eeeeeee eee 8e. Mee eeeeeeeee eeeeeeeee eeeeeee eee eeeee ee eee eeeeeee eeeee, eeeeeeee eee 8e, eee eeeeee eeee MMM. Mee eeeeeeeee eeeee eeeeeee eee eeeeeee eeee e eeee ee 88 (8 eeeee) ee e MMM/eMe8MMe eeee (8:8, 8 eeeee eMe8MMe eee eeee eeeee ee eeeeeeeee), eee eeee eeeeeeee eee 88e. Mee eeeeeee eeeee eee eeeeee eeee MMM, MM8Me8, eee eeeee eeeee eeeeeee eeeeeeee. Mee eeeeeee eee eeeeeeee eeeee MMMM eeeeeeeeeeee eee eeeeeeeeeeeeee; eeeee: 88%.

8-(8,8-Meeeeeeee-8-eeeeeeeeeee)eeeeeeeeeee-Meeeeeee Meeeeeee Meeeeeee eee Meeeeeeeeeee; Meeeeee Meeeeeeee:[‌888‌]

Meeeeeee: Meeeeeee eeee eeeeeeeee ee e 8:8 eeeee ee 888 eM Me8MM8 eeeeee (eM 8.8), ee eee eeeeeeee ee 88 eM eeee(8-eeeeeeeeeeee)eeeeeeeee, ee e eeeee eeeeeee eeeeeeeeeeeee ee 88 eM (eeeeeeeee 8888ee·eM8). Mee eeeee eM ee eee eeee eeeee eeeeeeee ee eee eeeeeeee eee 8.8. Mee eeeeeeee eee eeeeeeeee ee eeeeeeeeee MMMM. Meeeeee eeeeeeeeeeeeeee eeeeee eee eeeeeeeee eeee >88%. Me eee eee ee eee eeeeeeee eee eeeeeee eee eeeeeee eeee MMMM eeeeee (M8M/MMM 888:8) eee eee eeeeeee eee eeeeeeee ee eeeeeeeeeeeeeee MMMM; eeeee: eee eeeeeeee.

Meeeeeeeee eeeeeee ee eeeeeeeee 88: Meeeeee eeeeeeee eeee eeeeeeeee ee e eeeeeee ee MeMM/M8M (8:8) ee e eeeee eeeeeee eeeeeeeeeeeee ee eeeee 8.8ee·eM8 eee eee eeee eee eee ee e eeeeee eeee eee eeeeeeee ee eeeee eeeeeeee eee 88eee. Mee eeee eee eeeeeeeeee ee e Meeeeeee 888ee eeeeeeeeeeee eee eee eeeeeeee eee eeeeeeeee ee eeeeeeeeee MMMM. Me eee eee ee eee eeeeeeee eee eeee eee eeeeeee eeee MMMM eeeeee (M8M/MMM 888:8) eee eee eeeeeee eee eeeeeeee ee eeeeeeeeeeeeeee MMMM; eeeee: eee eeeeeeee.

References


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