Vitamin B12 umumiy sintezi - Vitamin B12 total synthesis

Ringning sintezi - A kashshofi

8-rasm: A-D-komponentlarning Garvard sintezi: halqa A

Ring A sintezining boshlang'ich nuqtasi metoksidimetil-indol edi H-1 tomonidan sintez qilingan kondensatsiya ning Shiff bazasi dan m-anizidin va asetoin. Bilan reaksiya Grignard reaktivi ning propargil yodid berdi rasemik propargil indolenin rac-H-2; halqaning yopilishi aminoketon rac-H-3 tomonidan olib kelingan BF₃ va HgO oraliq orqali MeOH da rac-H-2a (elektrofil a) ga majbur qilingan ikkita metil guruhi bilan cis- kinodik va termodinamik sabablarga ko'ra bog'liqlik.^[1]:521-522

9-rasm: A-D-komponentlarning Garvard sintezi: halqa rezolyutsiyasi

Qaror ikkiga rasemik aminoketon enantiomerlar. Reaktsiyasi rac-H-3 (-) bilan - etil izosiyanat tomonidan ruxsat berilgan izolyatsiya kristallanish ikkinchisidan biri diastereomerik karbamid hosilalari hosil bo'ldi (ikkinchisi kristallashmaydi). Ratsemik ketonni davolash rac-H-3 (yoki of.) ona likyorlari oldingi kristallanishdan) (+) - etil izosiyanat bilan birinchisining enantiomerini berdi karbamid lotin Pirolitik bu karbamid hosilalarining har birining parchalanishiga olib keldi enantiopure aminoketonlar, kerakli (+) - H-3va (-) - H-3.^[1]:524-525 "G'ayritabiiy" (-) - enantiomer (-) - H-3 mutlaqni aniqlash uchun ishlatilgan konfiguratsiya; turli keyingi bosqichlarda, (-) - H-3 va undan olingan enantio-oraliq moddalar qidiruv tajribalarida namunaviy birikmalar sifatida ishlatilgan.^[36]:173 Vudvord g'ayritabiiy enantiomer haqida yozgan "bizning tajribamiz shuni ko'rsatdiki, bu biz faqatgina ishonchli deb hisoblaydigan yagona namunaviy o'rganishdir".^[1]:529

10-rasm: A-D-komponentlarning Garvard sintezi: Ring A konfiguratsiyani aniqlash

Ni aniqlash mutlaq konfiguratsiya halqa-kashshofi (+) - H-3. Ushbu qat'iyat uchun aminoketonning levo-rotatsion ("g'ayritabiiy") enantiomeri (-) - H-3 qimmatbaho materiallarni tejash maqsadida ishlatilgan: amino guruhining asilatsiyasi (-) - H-3 bilan xloratsetil xlorid, keyinchalik mahsulotni davolash H-3a bilan kaliy t-utoksid yilda t-butanol, tetratsiklik keto-laktam H-3b. Uning keto karbonil metilen guruhiga aylantirildi desulfurizatsiya ning ditioketal ning H-3b bilan Raney nikeli bermoq laktam H-3c. Xushbo'y halqani yo'q qilish ozonoliz, o'z-o'zidan karboksil funktsiyasini yo'qotishni o'z ichiga oladi dekarboksilatsiya, bisiklik laktam-karboksilik kislotaga olib keldi H-3d. Ushbu material mahsulot bilan aniqlangan H-3 soat dan olingan (+) - kofur, formulada ko'rsatilgan bir xil konstitutsiya va mutlaq konfiguratsiyaga ega H-3d.^[1]:525-526

Ushbu identifikatsiyalash uchun material H-3d (+) - kofurdan quyidagi tarzda sintez qilingan: cis-izoketopin kislotasi H-3e, (+) - kofurdan adabiyotda tasvirlangan belgilangan yo'nalish bo'yicha olingan,^[68] korrespondent orqali konvertatsiya qilingan xlorid, azid va izosiyanat metilgauretan H-3f. Kaliy bilan davolashda t-utoksid t-butanol va keyinchalik KOH bilan, H-3f ga aylantirildi H-3 soat, aniq oraliq orqali H-3g. Ning ikkita namunasining kimligi H-3d va H-3 soat tasvirlangan ikkita yo'nalish bo'yicha olingan, ning mutlaq konfiguratsiyasini o'rnatgan (+) - H-3, halqaning enantiomeri - A kashshofi.^[1]:525-526

(-) kofurdan ring-D prekursorini sintezi

11-rasm: A-D-komponentlarning Garvard sintezi: D halqasi (-) dan kofur

(-) - kofur edi nitrozlangan berish uchun karbonil guruhining a-holatida oksim H-4, Bekman dekolti tegishli nitril amid bilan ta'minlanadi H-5. Hofman degradatsiyasi vositachi omin orqali va uning halqasining yopilishi laktamga olib keldi H-6. Uning konversiyasi N-nitroso lotin H-7 berdi diazo birikma H-8. Ning termal parchalanishi H-8 induktsiyalangan metil migratsiya siklopenten berish H-9. Kamaytirish H-10 (LiAlH₄ ), oksidlanish (xrom kislotasi ) aldegidga H-11, Wittig reaktsiyasi (karbometoksimetilenetrifenilfosforan ) ga H-12 va Ester guruhining gidrolizi nihoyat berdi trans-karbonat kislota H-13.^{[1]:527-528[14-eslatma]}

Ring-A va ring-D prekursorlarining "pentatsiklenon" bilan birikishi

12-rasm: A-D-tarkibiy qismlarining Garvard sintezi: A va D halqalarini "pentatsilenon" bilan biriktirish

N-trisiklik aminoketonning atsilatsiyasi (+) - H-3 xlorid bilan H-14 karboksilik kislota H-13 amid berdi H-15kaliy bilan davolashda t-utoksid t-butanol stereoelektiv ravishda pentatsiklik keto-laktam ishlab chiqarilgan H-16 orqali molekula ichi Mayklning reaktsiyasi ko'rsatilgan vodorod atomlarini trans aloqada bir-biriga yo'naltiradi. Kutish bilan Birchni kamaytirish ning xushbo'y halqa, ikkitasi uchun himoya guruhlari karbonil funktsiyalari ning H-16 talab qilingan, keton karbonil guruhi uchun bitta ketal H-17, ikkinchisi esa laktam karbonil yuqori sezgir enol eter H-20. Keyingisi himoya qilish davolash orqali erishildi H-17 bilan Meerwein tuzi (trietiloksonium tetrafluoroborat) bermoq iminium tuzi H-18, so'ngra ortoamidga o'tish H-19 (NaOMe / MeOH) va nihoyat toluolda isitib metanolning bitta molekulasini chiqarib tashlaydi. Birchni kamaytirish H-20 (lityum suyuqlikda ammiak, t-butanol, THF ) tetraen bilan ta'minlangan H-21. Diqqat bilan boshqariladigan sharoitda kislota bilan davolash birinchi navbatda oraliq mahsulotga olib keldi dione ga bog'langan β, γ holatidagi er-xotin bog'lanish bilan uyg'unlashgan dionadagi mavqei H-22, deb nomlangan pentatsiklenon.^{[1]:528-531[14]:5}

"Pentatsiklenon" dan "kornorsteron" gacha

13-rasm: A-D-komponentlarning Garvard sintezi: "pentatsilenon" dan "kornorsteron" gacha

Etilen ketal himoya guruhi pentatsiklenonda H-22 ning keton guruhiga aylantirildi H-23 kislota-katalizlangan gidroliz.^[1]:531 The dioksim birinchi navbatda diketon reaktsiyasi natijasida hosil bo'ladi H-23 bilan gidroksilammoniy xlorid edi regioselektiv ravishda gidrolizlangan (azot kislotasi / sirka kislotasi) kerakli mono-oksimga H-24. Bu steril ravishda ko'proq oksimdir xalaqit berdi Keton guruhi, uning azot atomi maqsad molekula halqasining azotiga aylanadi D. Buning uchun juda muhimdir konfiguratsiya monoksim er-xotin bog'lanishida, gidroksil guruhi steril ravishda kamroq to'siq qo'yilgan holatni egallaydi.^[1]:532 Siklopentenning ham, sikloheksenonning ham C, C juft bog'lari ichkariga kiradi H-24 keyin ajratilgan ozonoliz (MeOH da 80 ° C da ozon, davriy kislota ) va hosil bo'lgan karboksilik guruh bilan esterlangan CH₂N₂ ) diketonga H-25. An molekula ichi aldol kondensatsiyasi MeOH tarkibidagi 1,5-dikarbonil birligining pirrolidin asetat asos bo'lib, undan keyin tosilatsiya Oksim gidroksil guruhidan sikloheksenon hosilasini oldi H-26. Namlikdagi ikkinchi ozonoliz metil asetat, keyin davriy kislota va CH bilan davolash₂N₂ berdi H-27. Bekmanni qayta tashkil etish (MeOH, natriy polistirol sulfanat, 2 soat, 170 ° C) regioselektiv tarzda ishlab chiqarilgan^[1]:532 laktam H-27a (ajratilmagan) amin-karbonil kondensatsiyasida yana reaksiyaga kirishgan → tetrasiklga aldol kondensatsiya kaskadida H-28,^[1]:533-534 deb nomlangan a-kornorsteron, buni "burchak toshi"^[1]:534 kerakli A-D komponentining sintezida.^[1]:531-537

Ushbu birikma uni ochish uchun qattiq ishqoriy sharoitlarni talab qiladi laktam qo'ng'iroq, lekin u voyaga etmagan ekanligi aniqlandi izomer, shuningdek, reaktsiya aralashmasidan ajratilgan, b-kornorsteron H-29, ishqoriy sharoitda ushbu laktam halqasining ochilishini juda oson bajaradi.^[1]:536 Strukturaviy ravishda, ikkita izomer faqat propion kislotasi yon zanjirining A halqasida yo'nalishi bilan farq qiladi: b-izomer bu zanjirning laktam halqasi ochilgandan keyin hosil bo'lgan qo'shni sirka kislotasi zanjiriga nisbatan barqaror yo'naltirilganligiga ega. A-kornorsteronning muvozanati H-28 kuchli asosda isitiladi, so'ngra kislota va ishlov beriladi diazometan, sof b-kornorsteronning ajralib chiqishiga olib keldi H-29 90% hosilda.^[1]:537 Olti qo'shni to'g'ri mutlaq konfiguratsiya assimetrik markazlar g-kornorsteronda an tomonidan tasdiqlangan rentgen kristalining tuzilishini tahlil qilish bromo-b-kornorsteron^[69][1]:529 "g'ayritabiiy" konfiguratsiya bilan.^{[1]:538[14]:8[4](0:49:20-0:50:42)}

Propion kislotasi funktsiyasini D halqasida metoksikarbonil guruhi sifatida olib boradigan A-D-komponentni sintezi (A-D-komponentli model)

14-rasm: A-D-komponentlarning Garvard sintezi: f-differentsiyalanmagan model A-D-komponent

B-kornorsteronni davolash H-29 metanolik HCl bilan laktam halqasini ajratib oldi va hosil qildi enol eter hesperimine nomli lotin^[15-eslatma] H-30u. Aldegidga ozonoliz H-32u, aldegid guruhini kamaytirish NaBH₄ MeOH da birlamchi spirt H-33u va nihoyat, gidroksi guruhining mos keladigan konversiyasi mesilat bromidi berdi H-34u. Bu D halqasida farqlanmagan propionik kislota funktsiyasiga ega bo'lgan A-D-komponentli modelni tashkil etadi (ya'ni, boshqa barcha yon zanjirlar singari metil Ester guruhiga ega).^[1]:539-540

Propion kislota funktsiyasini D halqasida nitril guruhi sifatida olib boradigan A-D komponentining sintezi

15-rasm: A-D-komponentlarning Garvard sintezi: f-differentsial A-D-komponent

B-kornorsteronning konversiyasi H-29 tegishli A-D komponentiga H-34^[1]:538-539 a halqa D propion kislotasi yon zanjirining karboksil funktsiyasini o'z ichiga oladi nitril Boshqa barcha metoksikarbonil guruhlaridan ajralib turadigan guruh quyidagi bosqichlarni o'z ichiga oladi: davolash H-29 ning metanolik eritmasi bilan tiofenol va HCl fenil-tioenoleter hosilasini berdi H-30, ozonolizda past haroratda mos keladigan narsa tioester -aldegid H-31 va keyinchalik ammiak suyuq ammiak bilan ishlov berilganda H-32. Aldegid guruhini NaBH bilan kamaytirish₄ ga H-33, bilan birlamchi gidroksi guruhining mesilatsiyasi metansulfonik angidrid birlamchi amid guruhini kerakli nitril guruhiga aylantiradigan va nihoyat metansulfoniloksi guruhini A-D-komponentli bromid bilan almashtiradigan sharoitlarda H-34 propion kislota funktsiyasi bilan D halqasida nitril bo'lib, boshqa barcha yon zanjirlardan ajralib turadi.^{[1]:539-540[4](1:01:56-1:19:47)}

Ning qurilishi korin xromofor uning uchtasi bilan vinil amidin birliklar A va D halqalari orasidagi to'g'ridan-to'g'ri yagona bog'lanishdan tashqari, B vitaminini sintez qilishga urinish uchun asosiy muammo hisoblanadi.₁₂. B vitaminining umumiy sinteziga birinchi yondashuv₁₂ tomonidan boshlangan Kornfort^[43]:261-268 sintez qilingan halqa prekursorlarini birlashtirish vazifasiga duch kelganda to'xtatildi.^{[18]:1493,1496} Garvard A-D-komponentlarini ETH B-C-komponenti bilan birlashtirish keng ko'lamli izlanish ishlarini olib borishni talab qildi, ammo bu ETH modelida olingan bilimlarga qaramay, unchalik murakkab bo'lmagan (ya'ni kamroq periferiya bilan almashtirilgan) korinlarning sintezlarida. Rasmiy ravishda atigi ikkita C, C obligatsiyalarini tuzish uchun epik qo'shilish deb atash mumkin, 1967 yil boshidan beri davom etdi^[18]:1557 1970 yil iyunigacha.^[2]

ETH va Garvardda ham soddalashtirilgan ulanish bo'yicha keng qamrovli model tadqiqotlar enaminoid (halqa C) bo'lgan A-D komponentining analoglari imino- va miloddan avvalgi to'liq komponentning tio-iminoester hosilasi doimiy ravishda Garvard va ETH komponentlarini birlashtirishga oddiy korinlarni sintez qilishda juda muvaffaqiyatli bo'lgan usul bilan erishish mumkin emasligini, ya'ni molekulalararo enamino-imino (yoki tio-imino) esterining kondensatsiyasi^{[7][8][18]:1561[60]:41-58[1]:544[4](1:25:02-1:26:26)} Ushbu model tadqiqotlar natijasi Garvard A-D-komponentining yakuniy tuzilish turini aniqladi: C / D-muftasining tarkibiy qismi sifatida ishlashga qodir bo'lgan struktura. alkilatlovchi birikma orqali sulfidning qisqarishi,^{[8]:384-386[45]} ya'ni bromid H-34u.^{[7]:18-22[60]:47,51-52} Ushbu usul ETH guruhi tomonidan sintez qilishda allaqachon amalga oshirilgan edi B-C komponenti.^{[31]:16-19[35]:1927-1941[18]:1537-1540}

Optimal sharoitlarni izlash, birinchi navbatda A-D-komponentni ETH B-C-komponent bilan C / D-biriktirish uchun E-19, keyinchalik A-D-komponentli f-differentsiyalanmagan modelidan foydalanib, har ikkala laboratoriyada ham molekula ichidagi A / B-korin-rishtali yopilish holati kuzatildi.^[7-eslatma] H-34u^[1]:540 namuna sifatida.^{[2]:287-300[18]:1561-1564} Tomonidan ish natijasida Yoshito Kishi Garvardda,^{[2]:290[18]:1562[14]:11-12} va Piter Shnayder ETHda,^{[46]:12,22-29[18]:1563-1564} oxir-oqibat Garvardda C / D-kuplajining maqbul shartlari topildi, A va B halqalari orasidagi korin halqasini yopish uchun birinchi va eng ishonchli usul ETHda ishlab chiqildi.^[18]:1562 Ushbu model seriyasida ishlab chiqilgan C / D-ulanish va A / B-korinli halqalarni yopish protseduralari keyinchalik tegishli bosqichlarga tatbiq etildi. f-differentsial qatorlar kobirik kislota sintezining bir qismi sifatida.

Dicyano-kobalt (III) -5,15-bisnor-a, b, c, d, e, f, g-heptametil-kobirinat sintezi halqadan ajratilgan A-D-komponentli model

D / C ulanishi.^{[7]:22-23[2]:287-292[46]:12,22-28[18]:1561-1562}

Shakl 16: Garvard / ETH A / B kobirik kislotaga yondoshish: Garvardning A-D-komponentining ETH B-C-komponenti bilan D / C birikmasi

Ushbu bosqichdagi asosiy muammo asosiy ulanish mahsulotining labilligi edi, tioeter HE-35u, boshqa tioeterlarga izomerizatsiya qilish, dastlab qabul qilinadigan hosildorlik bilan takrorlanadigan protsedurada sulfidning qisqarishiga mos kelmaydi.^{[2]:287-290[4](1:26:59-1:32:00)} Kaliy tomonidan qo'zg'atilgan t- THF tarkibidagi butoksid /t-butanol qat'iy nazorat ostida bo'lgan sharoitda havo va namlikni qat'iyan chiqarib tashlagan holda, A-D-komponentli model H-34u B-C-komponenti bilan silliq reaksiyaga kirishdi E-19^[46]:53-58 oltingugurt ko'prigi bilan biriktiruvchi mahsulotni berish HE-35u, "tioeter I tip" deb nomlangan bo'lib, asosan miqdoriy rentabellikga ega.^[2]:287-288 Shu bilan birga, ushbu mahsulotni faqat juda ehtiyotkorlik bilan boshqariladigan sharoitda ajratib olish mumkin edi, chunki u juda oson izomerik tioetergacha juda osonlik bilan muvozanatlashadi (masalan, xromatografiya yoki metileneklorid eritmasidagi trifloroasetik kislota izlari). HE-36u Tioeter I tipidan farqli o'laroq konjugativ stabillashgan vinil amidinning b-tizimini o'z ichiga olgan (tioeter II tip).^[2]:289 Shartlarga qarab, yana bir izomer HE-37u (thiother III turi) kuzatildi.^[2]:290 ETH mahsulotlarini birlashtiradigan bunday aralashmalaridan boshlab har xil sharoitlarda (masalan, metil-simob kompleksi, BF₃, trifenilfosfin^{[46]:58-65[2]:291}) ni keltirib chiqarishi aniqlandi (orqali HE-38u) ga qisqarish bosqichi HE-39u in moderate yields.^{[18]:1562[2]:287-292} With the choice of the solvent found to be crucial,^{[4](1:34:52-1:35:12)} the optimal procedure at Harvard was heating thiother type II HE-36u yilda sulfolan in the presence of 5.3 equivalents trifloroasetik kislota and 4.5 equivalents of tris-(β-cyanoethyl)-phosphine at 60 °C for 20 hours, producing HE-39u in up to 85% yield.^{[2]:292[46]:65-72} Later it was discovered that nitrometan could also be used as solvent.^{[4](1:34:52-1:35:13)[46]:28}

A/B-ring closure.^{[2]:293-300[46]:12,29-39[18]:1562-1564}

The problem of corrin-ring closure between rings A and B was solved in two different ways, one developed at ETH, the other pursued at Harvard.^[30]:19 Both methods correspond to procedures developed before in the synthesis of metal complexes^[70] as well as free ligands^[71] of simpler corrins.^{[7]:25-28[8]:387-389[18]:1563} In the explorations of ring-closure procedures for the much more highly substituted A/B-seco-corrinoid intermediate HE-39u, the ETH group focused on the intramolecular version of the oxidative sulfide contraction method, eventually leading to the dicyano-cobalt(III)-complex HE48u.^{[46]:29-39[2]:297-299} This first totally synthetic corrinoid intermediate was identified with a corresponding sample derived from vitamin B₁₂.^[18]:1563

At Harvard, it was shown that the closure to the corrin macrocycle could also be realized by the method of thioiminoester/enamine condensation.^[2]:299-300 All reactions described here had to be executed on a very small scale, with "... the utmost rigour in the exclusion of oxygen from the reaction mixtures"^[2]:296, and most of them also under strict exclusion of moisture and light, demanding very high standards of experimental expertise.^[2]:304

The major obstacle in achieving an A/B-corrin-ring closure was the exposure of the highly unstable ring B ekzosiklik methylidene double bond, which tends to isomerize into a more stable, unreactive endocyclic position with great ease.^{[46]:86,97-98[2]:293-294[3]:161[18]:1562}

Figure 17: Harvard/ETH A/B approach to cobyric acid: A/B-ring closure to the f-undifferentiated model 5,15-bisnorcobyrinat

The problem was solved at ETH^{[18]:1562-1563[46]:29-39,126-135} by finding that treatment of the thiolactone-thiolactam intermediate HE-40u (obtained from HE-39u by reacting with P₂S₅^[46]:73-83) bilan dimetilamin in dry MeOH (room temperature, exclusion of air and light) smoothly opens the tiolakton ring at ring B, forming by elimination of H₂S the exocyclic methylidene double bond as well as a dimethylamino-amide group in the acetic acid side chain.^{[46]:32-34,96-99} These conditions are mild enough to prevent double bond tautomerization to the thermodynamically more stable isomeric position in the ring. Immediate conversion with a Zn-perchlorate-hexa(dimethylformamide) complex in methanol to zinc complex HE-41u, followed by oxidative coupling (0,05 mM solution of Men₂ /KI in MeOH, 3 h) afforded HE-42u.^[46]:100-105 Sulfide contraction (triphenylphosphine, trifluoroacetic acid, 85 °C, exclusion of air and light) followed by re-complexation with Zn(ClO₄)₂ (KCl, MeOH, diisopropylamine ) led to the chloro-zinc complex HE-43u.^[46]:105-116 The free corrinium salt formed when HE-43u was treated with trifluoroacetic acid in asetonitril was re-complexed with anhydrous CoCl₂ in THF to the dicyano-cobalt(III)-complex HE-44u.^{[46]:117-125[2]:295} Conversion of the dimethylamino-amide group in the acetic acid side chain of ring B into the corresponding methylester group (O-methylation by trimethyloxonium tetrafluoroborate, followed by decomposition of the iminium salt with aqueous NaHCO₃) afforded totally synthetic 5,15-bisnor-heptamethyl cobyrinate HE-48u.^{[46]:11,117-125} A crystalline sample of HE-48u was identified via UV/VIS, IQ va ORD spectra with a corresponding crystalline sample derived from vitamin B₁₂^{[46]:42,135-141[53]:14,64-71,78-90[2]:287,301-303[3]:146-150[72]}

Later at Harvard,^[2]:299-300 the A/B-corrin-ring closure was also achieved by converting the thiolactone-thiolactame intermediate HE-40u to thiolactone-thioiminoester HE-45u tomonidan S-methylation of the thiolactam sulfur (MeHgOi-Pr, then trimethyloxonium tetrafluoroborate). Mahsulot HE-45u was subjected to treatment with dimethylamine (as in the ETH variant), forming the highly labile methylidene derivative HE-46u, which then was converted with anhydrous CoCl₂ in THF to dicyano-cobalt(III) complex HE-47u, the substrate ready to undergo the (A⇒B)-ring closure by a thioiminoester/enamine condensation. A careful search at Harvard for reaction conditions led to a procedure (KO-t-Bu, 120 °C, two weeks) that gave corrin Co complex HE-44u, identical with and in overall yields comparable with HE-44u obtained by the ETH variant of the sulfide contraction procedure.^[2]:300 Since in corrin model syntheses such a C,C-condensation required induction by a strong base, its application in a substrate containing seven methylester groups was not without problems;^[18]:1562 in a, milder reactions conditions were applied.^[3]:162

Synthesis of dicyano-cobalt(III)-5,15-bisnor-a,b,d,e,g-pentamethyl-cobyrinate-c-N, N-dimethylamide-f-nitrile (the common corrinoid intermediate) from the ring-D-differentiated A-D-component

Figure 18: Harvard/ETH A/B approach to cobyric acid: coupling of the Harvard f-differentiated A-D-component with the ETH B-C-component to the common corrinoid intermediate

The A-D-component H-34^[8-eslatma] with its propionic acid function at ring D differentiated from all the other carboxyl functions as nitrile group had become available at Harvard in spring 1971.^[49]:23 As a result of the comprehensive exploratory work that had been done with the model A-D-component at Harvard and ETH,^{[2]:288-292[46]:22-28[18]:1561-1562} joining the proper A-D-component H-34 with the B-C-component E-19 by three operations H-34 + E-19 →→ HE-36 → HE-39.^{[3]:158-159[4](1:19:48-1:36:15)}

Closing the corrin ring was achieved in the sequence HE-39 (P₂S₅, ksilen, γ-picoline )→ HE-40^{[4](1:36:45-1:37:49)} → HE-41^{[4](1:37:51-1:42:33)} → HE-42^{[4](1:42:35-1:44:34)} → HE-43 (overall yield "about 60 %"^{[4](1:44:35-1:46:32)}), and finally to cobalt complex HE-44.^{[4](1:46:34-1:52:51)[3]:160-166} Reactions in this sequence were based on the procedures developed in the undifferentiated model series.^{[2]:293-300[46]:29-39[18]:1562-1564} Two methods were available for the A/B-ring closure: oxidative sulfide contraction within a zinc complex, followed by exchange of zinc by cobalt (ETH^[3]:162-165), or the Harvard alkylative variant of a sulfide contraction,^[3]:160-162 thio-iminoester /amin condensation of the cobalt complex (improved reaction conditions: diazabicyclononanone in DMF, 60 °C, several hours^[3]:162). Woodward preferred the former one:^[3]:165 "...the oxidative method is somewhat superior, in that it is relatively easier to reproduce, .... ".^{[4](1:52:37-1:53:06)}

The corrin complex dicyano-cobalt(III)-5,15-bisnor-pentamethyl-cobyrinate-c-N, N-dimethylamide-f-nitrile HE-44 took up the role of the common corrinoid intermediate in the two approaches to cobyric acid synthesis: HE-44 ≡ E-37. Due to the high configurational lability of C-H chirogenic centers C-3, C-8 and C-13^{[4](1:21:49-1:23:42,1:35:43-1:36:14,1:51:51-1:52:30)} da ligand periphery in basic or acidic milieu, separation by HPLC was indispensable for isolation, purification and characterization of pure diastereomers of this and the following corrinoid intermediates.^{[3]:165-166[9]:88-89[4](1:53:07-2:01:24)}

Figure 19: Harvard preparation of the ring-C precursor from (+)-camphor

Starting material for the synthesis of a ring-C precursor was (+)-camphorquinone H-35^[16-eslatma] which was converted to the acetoxy-trimethylcyclohexene-carboxylic acid H-36 tomonidan BF₃ yilda sirka angidrid, a reaction pioneered by Manasse & Samuel in 1902,^[73], already successfully applied in a previous synthesis of the ring-C precursor by Pelter and Cornforth.^[6-eslatma] Conversion of H-36 to amide H-37 was followed by its ozonoliz ga peroksid H-38 which was reduced to the keto-süksinimid H-46 by zinc and MeOH. Treatment with methanolic HCl gave lactam H-40, followed by thermal yo'q qilish of methanol to the ring-C precursor H-41^{[1]:540-542[46]:49-50[14]:4-5,15} This was found to be identical with the ring-C precursor E-13 prepared by a different route^[5-eslatma] at ETH.^{[59]:32[42]:30,33-34,81}

Syntheses of the ring-B precursor

Two syntheses of ring-B precursor (+)-E-5 were realized; the one starting from 2-butanone was used further.^[6]:188 Two pathways for the conversion of the ring-B precursor into the ring-C precursor (+)-E-5 → (−)-E-13 ≡ H-41 were developed, one at ETH,^{[42]:15-39[1]:544}, and one at Harvard.^{[6]:193[17-eslatma]} These conversions turned out to be inadequate for producing large amounts of ring-C-precursor.^{[44]:38[18]:1561} However, the pathway developed at ETH served the purpose of determining the absolute configuration of the ring-B precursor.^{[6]:193[59]:32} Bulk amounts of ring-C precursor to be used for the production of the B-C-component at ETH^{[42]:40[6]:193[31]:631} were prepared at Garvard dan (+)-camphor by a route originally developed by Pelter and Kornfort.^[6-eslatma]

Figure 20: ETH synthesis of the B-C-component: synthesis of the two enantiomers of the ring-B precursor

Ring-B precursor from 2-butanone and glyoxylic acid. Aldol kondensatsiyasi o'rtasida 2-butanone va glyoksilik kislota by treatment with concentrated fosfor kislotasi ) gave stereoselectively (trans)-3-methyl-4-oxo-2-pentenoic acid E-1.^{[37]:11-20,45-45} Diels-Alder reaktsiyasi E-1 bilan butadien in benzene in the presence of SnCl₄ afforded the rasemate ning chiral Diels-Alder adduct E-2 qaysi edi hal qilindi into the enantiomers by sequential salt formation with both (−)- and (+)-1-phenylethylamine.^{[41]:22,59-62} The chirogenic centers of the (+)-enantiomer (+)-E-2 possessed the absolute konfiguratsiya ning ring B in vitamin B₁₂.^{[58]:35[6]:191} Oxidation of this (+)-enantiomer with xrom kislotasi in acetone in the presence of sulfat kislota afforded the dilactone (+)-E-3 of the intermediary tricarboxylic acid E-3a.^{[41]:35,72-73} Thermodynamic control of dilactone formation leads to the cis-configuration of the ring junction.^[41]:32-34 Elongation of the acetic acid side chain of (+)-E-3 tomonidan Arndt-Eistert reaction (via the corresponding kislota xloridi and diazoketone) gave dilactone (+)-E-4.^{[59]:15-16,65-67} Davolash (+)-E-4 bilan NH₃ in MeOH at room temperature formed a dual mixture of isomeric laktam -laktonlar in a ratio of 2:1, with ring-B precursor (+)-E-5 predominating (isolated in 55% yield).^{[44]:12-17,57-63[6]:186-188[12][1]:542-543} The isomeric lactam-lactone could be isomerized to (+)-E-5 by treatment in methanolic HCl.^{[59]:24-26,81-84}

Figure 21: ETH synthesis of the B-C-component: Alternative Synthesis of the (racemic) Ring-B Precursor (only one enantiomer shown for racemates)

Alternative synthesis of rasemik ring-B precursor from Hagemann's ester: implementation of the amidacetal-Claisen rearrangement. Five steps were needed to transform Hagemann's ester rac-E-6 into the racemate of the lactam-lactone rac-E-5 form of the ring-B precursor.^{[58]:14-31[6]:188-190} The product of the C-methylation step rac-E-6 → rac-E-7 (NaH, CH₃Men ) was purified via its crystalline oksim. The cis-hydroxy-ester (configuration secured by lactone formation^[58]:64) resulting from the reduction step rac-E-7 → rac-E-8 (NaBH₄ ) had to be separated from the trans izomer. Termal rearrangement rac-E-8 → rac-E-9 tashkil etadi amalga oshirish ning amidacetal-Claisen rearrangement in organic synthesis,^{[74][58]:36-49} a precedent to Johnson's orthoester-Claisen va Ireland's ester-enolate rearrangement.^[75] Ozonoliz (O₃ /MeOH, HCOOH /H₂O₂ ) ning N, N-dimethylamide ester rac-E-9 afforded dilactone acid rac-E-10, from which two reactions led to lactam-lactone methylester rac-E-7, the racemate of ring-B precursor (+)-E-7.^[58]:57-67

Determination of absolute configuration of (+)-ring-B precursor via its conversion into the (+)-ring-C precursor

Figure 22: ETH synthesis of the B-C-component: Conversion of Ring-B Precursor to Ring-C Precursor

The conversion of ring-B precursor into the ring-C precursor was based on a reductive dekarbonillanish ning tiolakton E-12 with chloro-tris-(triphenylphosphino)-rhodium(I).^{[42]:14-32[6]:191-193[12]} Treatment of a methanolic solution of ring-B precursor (+)-E-5 with diazomethane in the presence of katalitik amounts of natriy metoksid, followed by thermal yo'q qilish of methanol, gave methylidene lactam E-11, which was converted to the thiolactone E-12 with liquid H₂S containing a catalytic amount of trifluoracetic acid.^{[42]:15-16,56-58} Isitish E-12 in toluene with the Rh(I)-complex afforded ring-C precursor (−)-E-13 besides the corresponding cyclopropane derivative E-14. Ring-C precursors prepared via this route and from (+)-camphor at Harvard ^[1]:540-542 were found to be identical: (−)-E-13 ≡ H-41.^[42]:33-34

Ozonolysis of ring-C precursor (−)-E-13 berdi süksinimid lotin (−)-E-15.^{[42]:33-35,88-89} This succinimide was found to be identical^{[6]:193[1]:543-544} in constitution and optik aylanish (i.e., configuration) with the corresponding succinimide derived from ring C of Vitamin B₁₂, isolated after ozonolysis of crystalline heptamethyl-cobyrinate (cobester^[9-eslatma]) prepared from Vitamin B₁₂.^{[54]:9-18,67-70}

The approach pursued at Harvard for conversion of ring-B precursor into ring-C precursor was based on a fotokimyoviy degradation of the acetic acid side chain carboxyl group, starting from (+)-E-7 prepared at ETH.^[17-eslatma]

Coupling of ring-B and ring-C precursors to the B-C-component. Implementation of the sulfide contraction C,C-condensation method

The iminoester /enamine C,C-kondensatsiya method for constructing the vinylogous amidine system, developed in the model studies on korin sintez,^[26][33] failed completely in attempts to create the targeted C,C-bond between ring-B precursor (+)-E-5 with ring-C precursor (−)-E-13 to give the B-C-component E-18.^{[6]:193-194[8]:379[1]:544} The problem was solved by "intramolecularization" of the bond formation process between the elektrofil (thio)iminoester carbon and the nukleofil methylidene carbon of the amin system through first oxidatively connecting these two centers by a sulfur bridge, and then achieving the C,C-bond formation by a now intramolecular thio-iminoester/enamine condensation with concomitant transfer of the sulfur to a thiophile.^{[6]:194-197[8]:380-386[18]:1537-1538}

Figure 23: ETH synthesis of the B-C-component: coupling of the ring B and C precursors (implementation of C/C-coupling by the sulfide-contraction method)

Conversion of lactam (+)-E-5 into the corresponding thiolactam E-16 (P₂S₅),^{[44]:20-23,74-75} oxidation of E-16 bilan benzoil peroksid in the presence of ring-C precursor (−)-E-13 (prepared at Harvard by the Cornforth route^[6-eslatma]), followed by heating the reaction product E-17 yilda triethylphosphite (as both solvent and thiophile) afforded B-C-component E-18 as a (not separated) mixture of two epimers (regarding the configuration of the propionic side chain at ring B) in up to 80 % yield.^{[44]:38-43,96-102[31]:16-19[8]:381-383[46]:20-21,50-52}

The bracketed formulae in the reaction scheme illustrate the type of mexanizm operating in the process: E-16a = primary coupling of E-12 va E-10 ga E-13; E-17a = extrusion of the sulfur atom (captured by thiophile) to E-14, where it is left open whether this latter process occurs at the stage of the episulfide. This reaction concept developed at this stage, dubbed sulfide contraction,^{[6]:199[45][18]:1534-1541[35]:1927-1941} turned out to make possible the construction of all three meso-carbon bridges of the vitamin's corrin ligand in both approaches of the synthesis.^{[12][11][2]:288-292,297-300[3]:158-164}

The conversion of bicyclic lactone-lactam E-18 into the corresponding thiolactone-thiolactam E-20 was brought about by heating with P₂S₅ /4-methylpyridine yilda ksilen at 130 °C; milder condition produced thiolactam-lactone E-19uchun ishlatilgan birlashma with the Harvard A-D-components.^[49]:73-83

Synthesis of ring-D precursor for the A/D approach

Figure 24: ETH A/D approach to cobyric acid: synthesis of ring-D precursor

The starting material for the ring-D precursor,^{[59]:40-61[61]:17-22[12]} the (−)-enantiomer of the dilactone-carboxylic acid (−)-E-3, was prepared from the (−)-enantiomer of the Diels-Alder qo'shib qo'yish (−)-E-2^[18-eslatma] by oxydation with xrom kislotasi /sulfuric acid in acetone.^{[41]:35,72-73} Davolash (−)-E-3 with NH₃ in MeOH gave a lactone-lactam-acid which was esterified with diazometan to the ester E-21,^[59]:104-110 the lactone ring of which was opened with KCN in MeOH to give E-22.^[59]:114-116 Conventional conditions of an Arndt-Eistert reaction (SOCl₂: acid chloride, then CH₂N₂ in THF: diazoketone, treated with Ag₂O in MeOH) led to an – unforeseen, yet useful – ring closure of the originally formed chain-elongated ester through participation of the cyano group as a neighboring elektrofil, affording the bicyclic enamino-ester derivative E-23.^[59]:116-120 Hydrolysis with aqueous HCl, accompanied by decarboxylation, and re-esterification with diazomethane gave keto-lactam-ester E-24.^{[59]:123-126[61]:40-41} Ketalization ((CH₂OH)₂, CH(OCH₃)₃, TsOH ) ning E-24 and conversion of this lactam-ester to thiolactam E-25 (P₂S₅ ) was followed by reductive removal of the sulfur with Raney nikeli, atsetilatsiya of the amino group, and hydrolysis of the ketal (AcOH) to afford E-26.^[61]:42-59 This was converted by deacetylation of the amino group with HCl, and then by treatment with NH₂OH/HCl, MeOH/NaOAc into oxime E-27. Beckmann fragmentation (HCl, SOCl₂ in CHCl₃, N-polystyryl-piperidine) of this oxime E-27 produced imino-nitrile E-28,^[61]:60-67 which, when treated with brom (in MeOH, phosphate bufer pH 7.5, -10 °C) gave ring-D precursor E-29.^[49]:84-88

Conversion of the ring-B precursor into the ring-A precursor for the A/D approach

Figure 25: ETH A/D approach to cobyric acid: conversion of ring-B precursor into ring-A precursor

The ring-A precursor (−)-E-31 required in the A/D approach is a close derivative of ring-B precursor (+)-E-5. Its preparation from (+)-E-5 required opening of the lactone group (KCN in MeOH), followed by re-esterification with diazomethane to E-30, then conversion of the lactam group into a thiolactam group with P₂S₅ hosil bermoq (−)-E-31.^{[49]:63-72[12]}

Coupling of the B-C-component with ring-D and ring-A precursors

The most efficient way of attaching the two rings D and A to the B-C-component E-18 was to convert E-18 directly into its thiolactam-tiolakton lotin E-20 and then to proceed by first coupling ring-D precursor E-29 to ring C, and then ring-A precursor E-31 to ring B, both by the sulfide contraction method.^{[49]:26-31[9]:80-83[12]} The search for the reaction conditions for these attachments was greatly facilitated by exploratory work done on the two sulfide contraction steps in the A/B approach model study.^{[49]:27[46]:22-39[2]:285-300}

Figure 26: ETH A/D approach to cobyric acid: Attaching ring-C and ring-A precursors to the B-C-component to yield the A/D-seco-corrin

Attachment of ring-D precursor E-29 to the ring-C thiolactam in E-20 by sulfide contraction via alkylative coupling (t-BuOK in t-BuOH/THF, tris-(β-cyano-ethyl)-phosphin/CF₃COOH yilda sulfolan ) afforded the B/C/D-sesqui-corrinoid E-32.^[49]:89-97 To attach ring-A precursor E-31, the ring B of E-32 was induced to expose its ekzosiklik metiliden qo'shaloq bog'lanish by treatment with dimetilamin in MeOH (using the method^[19-eslatma] developed by Schneider^[46]:32-34) forming E-33^[49]:108-115 which was subjected to the following cascade of operations:^[49]:130-150 iodination (N-iodosuksinimid, CH₂Cl₂, 0°), coupling with the thiolactam sulfur of the ring-A precursor E-31 [(CH₃)₃Si]₂N-Na in benzene/t-BuOH), complexation (Cd(ClO₄)₂ in MeOH), treatment with trifenilfosfin /CF₃COOH in boiling benzene (sulfide contraction) and, finally, re-complexation with Cd(ClO₄)₂/N, N-diisopropylethylamine in benzene/MeOH). These six operations, all carried out without isolation of oraliq mahsulotlar, gave A/D-seco-corrin complex E-34 as mixture of peripheral epimers (separable via HPLC^[49]:143-147) in 42-46 % overall yield.^[49]:139

A/D-corrin-ring closure by the photochemical A/D-seco-corrin→corrin cycloisomerization to dicyano-cobalt(III)-5,15-bisnor-a,b,d,e,g-pentamethyl-cobyrinate-c-N, N-dimethylamide-f-nitrile (the common corrinoid intermediate)

The conditions and prerequisites for the final (A⇒D)-korin -ring closure were taken over from extensive corrin model studies.^{[34][76][9]:71-74,83-84[18]:1565-1566[35]:1942-1962} Problems specific to the cobyric acid synthesis that had to be tackled were:^[9]:84-88 the possible formation of two diastereomerik A/D-trans-junctions in the ring closure,^[49]:37-38 exposure of the methylidene double bond at ring A of the A/D-seco-corrin E-34 in a labile Cd complex,^{[49]:35-36[18]:1566} and epimerizability of the peripheral stereogenic centers C-3, C-8 and C-13 before and after ring closure.^{[49]:39[3]:148-150}

Figure 27: ETH A/D approach to cobyric acid: photochemical A/D-seco-corrin→corrin cycloisomerization to the common corrinoid intermediate

In the application of this novel process in the A/D approach of the cobyric acid synthesis,^{[9]:86-95[49]:39-53[12]:1419} the reaction proceeded most efficiently and with highest lasan stereoelektivlik in favor of the natural A/D-trans junction in an A/D-seco-corrin cadmium complex.^{[49]:42-45[3]:166} Treatment of Cd-complex E-34 as mixture of peripheral epimers bilan 1,8-Diazabitsiklo (5.4.0) undec-7-ene yilda sulfolan at 60 °C under strict protection against light to yo'q qilish the cyano group at ring A, directly followed by re-treatment with Cd(ClO₄)₂, led to labile^[49]:172 A/D-seco-corrin complex E-35 as a mixture of peripheral epimers. This was directly subjected to the key step, the photochemical ring closure reaction under rigorous exclusion of air:^[49]:40 visible light, under Argon, MeOH, AcOH, 60° C. Product of the A/D-ring closure was the free corrin ligand E-36, as the originally formed Cd-corrinate – in contrast to the Cd-seko-corrinate E-35  – decomplexes in the reaction medium.^{[49]:173[12]:1419} Korrin E-36 was immediately complexed (CoCl₂,^{[18]:1499-1500,1563-64} KCN, air, H₂O, CH₂Cl₂) and finally isolated (thick-layer xromatografiya ) as mixture of peripheral epimers in 45-50 % yield over four operations:^[49]:169-179 the common corrinoid intermediate dicyano-cobalt(III)-complex E-37 ≡ HE-44.^[20-eslatma]

HPLC analysis of this mixture E-37 showed the presence of six epimers with natural ligand merosxo'rlik (Σ 95%, CD spectra ), among them 26% of natural diastereomer 3α,8α,13α, and an equal amount of its C-13 neo-epimer 3α,8α,13β.^{[49]:46,179-186[12]:1414} Two HPLC fractions (Σ 5%) contained diastereomers with unnatural ligand helicity, as shown by inverse CD spectra.^[49]:42-43 Product mixtures from several such cycloisomerizations were combined for preparative HPLC separation and full characterization of the 14 isolated diastereomers of E-37^[49]:207-251 (of 16 theoretically possible, regarding helicity and the epimeric centers C-3, C-8, C-13^[49]:39).

Figure 28: ETH A/D approach to cobyric acid: coil selectivity in A/D-ring closure

In an analytical run, the mixture of cadmium-seco-complex epimers E-35 was separated by HPLC (in the dark) into the natural chloro-cadmium-3α,8α,13α-A/D-seco-corrinate diastereomer (ααα)-E-35 and four other epimer fractions^[49]:281-293 Ustiga nurlanish^[49]:53[12] and following cobaltation, (ααα)-E-35 ishlab chiqarilgan E-37 in yields of 70-80% as an essentially dual mixture of mainly the 3α,8α,13α epimer, besides some 3α,8α,13β epimer. Less than 1% of fractions with unnatural coil were formed (HPLC, UV/VIS, CD ).^[49]:293-300

Mechanistically, fotokimyoviy A/D-seco-corrin corrin cycloisomerization involves an antarafasiyal sigmatropic shift of the α-hydrogen of the CH₂ position C-19 at ring D to the CH₂ position of the methylidene group at ring A within a uchlik hayajonlangan holat, creating a transient 15-center-16-electron π-system (see E-35a yilda Anjir. 27 ) that antarafacially collapses between positions C-1 and C-19 to the corrin system.^{[34][35]:1946,1967-1993[77]} The coil selectivity of the ring closure in favor of the corrin ligand's natural helicity is interpreted as relating to the difference in sterik to'siq between the g-methoxycarbonyl acetic acid chain at ring D and the methylidene region of ring A in the two possible helical coil configurations of the A/D-seco-corrin complex (fig. 28).^{[49]:38[35]:1960-1962}

Figure 29: ETH/Harvard joint final steps: Introduction of methyl groups at the meso positions C-5 and C-15

This introduction of methyl groups could draw on exploratory studies on model corrins^{[7]:13-14[8]:375-377[78][18]:1528,1530-1532} as well as on exploratory experiments carried out at ETH on cobester^[9-eslatma] and its (c→C-8)-lactone derivative.^[53]:27-43 Chloromethyl benzyl ether alkylated the meso position C-10 of cobester, but not that of the corresponding lakton, the difference in behavior reflecting the difference in sterik to'siq exerted on the meso position C-10 by its neighboring substituents.^[53]:37-39 This finding was decisive for the choice of the substrate to be used for introducing methyl groups at meso positions C-5 and C-10 of E-37/HE-44.^{[9]:96-99[53]:19[3]:167[18]:1567-1568} In this final phase of the synthesis, HPLC again turned out to be absolutely indispensable for separation, isolation, characterization and, above all, identification of pure isomers of dicyano-cobalt(III)-complexes of totally as well as partially synthetic origin.^{[9]:96-102[3]:165[53]:61-63[5](0:21:13-0:25:28)[18]:1566-1567}

The first step was to convert the c-N, N-dimethylcarboxamide group of E-37/HE-44 into the (c→C-8)-lactone derivative E-38/HE-45 by treatment with yod /AcOH effecting iodination at C-8, followed by intramolecular O-alkylation of the carboxamide group to an iminium salt that hydrolyses to the lactone.^{[61]:23,90-108[3]:166-167[4](2:02:18-2:09:02)} This lactonization leads to cis-fused rings.^{[53]:19[5](0:09:34-0:10:43)} Reaction of (c→C-8)-lactone E-38/HE-45 with chloromethyl benzyl ether in asetonitril in the presence of LiCl gave, besides mono-adduct, the bis-benzyloxy adduct E-39/HE-46. Davolashda tiofenol, this produced the bis-phenylthio-derivative E-40/HE-47. Bilan davolash Raney nikeli in MeOH not only set free the two methyl groups at the meso positions, but also reductively opened the lactone ring to the free c-carboxyl group at ring B, producing the correct α-konfiguratsiya at C-8. Esterification of c-carboxyl with diazometan afforded hexamethylester-f-nitrile E-41/HE-48.^{[53]:19-21,39-43,146-205[3]:167-169} For steric reasons, only the predominant^{[53]:19[61]:24[4](2:08:20-2:09:02)} C-3 α-epimer (with the C-3 side chain below the plane of the corrin ring) reacted to a 5,15-disubstituted mahsulot E-38/H-45, the reaction thus amounting to a chemical separation of the C-3 epimers.^{[53]:40[5](0:12:51-0:14:33,0:15:56-0:16:24)}

In improved procedures developed at Harvard later in 1972,^{[18]:1569 footnote 62} the reagent chloromethyl benzyl ether was replaced by formaldegid /sulfolane/HCl in acetonitrile for the alkylation step, and Raney nickel in the kamaytirish step was replaced by zinc/acetic acid to give E-41/HE-48.^{[5](0:00:32-0:21:12)}

Figure 30: ETH/Harvard joint final steps: hexamethylcobyrinate-f-amide (synthesis and identification) to cobyric acid

Concentrated H₂SO₄ at room temperature converted the nitrile function of pure (3α,8α,13α)-E-41/HE-48 into the primary f-amid guruhi E-42/HE-49, besides partial epimerization at C-13;^{[9]:100-103[53]:21,134-136[3]:150-151,169-170} an alternative procedure for the selective f-nitrile→f-amide conversion (BF₃ in CH₃COOH) later developed at Harvard proceeded without epimerizatsiya at C-13.^{[18]:1569 footnote 62[5](0:46:40-0:49:45)[53]:21} A crystalline sample of the 3α,8α,13α-epimer of dicyano-cobalt (III)-a,b,c,d,e,g-hexamethyl-cobyrinate-f-amide E-42/HE-49, isolated by HPLC, was the first totally synthetic intermediate to be chromatographically and spektroskopik jihatdan identified with a relay sample made from vitamin B₁₂.^{[53]:136-141[3]:170}

In the remaining steps of the synthesis, only epimerization at C-13 played an important role,^[53]:19-21 with 13α being the configuration of the natural corrinoids, and 13β known as neo-epimers of vitamin B₁₂ and its derivatives;^{[3]:169-170[79]} these are readily separable by HPLC.^{[5](0:19:30-0:20:21)[53]:135,208-209}

In the course of 1972, comprehensive identifications (HPLC, UV/VIS, IQ, NMR, CD, mass spectra ) of crystalline samples of totally synthetic intermediates with the corresponding compounds derived from vitamin B₁₂ were carried out in both laboratories: individually compared and identified were the 3α,8α,13α and 3α,8α,13β neo-epimer of f-amide E-42/HE-49, as well as the corresponding pair of C-13-epimeric nitriles E-41/HE-48.^{[53]:206-221[55]:46-47[5](0:27:28-0:46:32)} All these dicyano-cobalt(III)-complexes are soluble in organic solvents^[54]:11 in which the separation power of HPLC by far exceeds that of analytical methods operating in water,^[53]:44-45 the solvent in which cobyric acid was to be identified, and where it exists as two easily equilibrating aquo-cyano complexes, epimeric regarding the position of the two non-identical axial Co ligandlar.^{[61]:196-197[55]:49-60}

These thorough identifications of the totally synthetic with partially synthetic materials mark the accomplishment of the two syntheses. They also reciprocally provided structure proof for a specific constitutional isomer isolated from a mixture of isomeric mono-amides formed in the partial ammonolysis of the B₁₂-derived cobester,^[9-eslatma] tentatively assigned to be the 3α,8α,13α-f-amide E-42/HE-49 (see fig. 30).^{[54]:9-18,67-70[53]:226-239[57]}

The final task of reaching cobyric acid from f-amide E-42/HE-49 required the critical step of hydrolysing the singular amide function into a free carboxyl function without touching any of the six methoxycarbonyl groups around the molecule's periphery. Since exploratory attempts by the conventional method of amide hydrolysis via nitrozlash led to detrimental side reactions at the xromofor, a novel way of "hydrolysing " the f-amide group without touching the six methylester groups was conceived and explored at ETH: treatment of f-amide E-42/HE-49 (B.₁₂-derived relay material) with the unusual reagent α-chloro-propyl-(N-cyclohexyl)-nitrone^[80] va AgBF₄ in CH₂Cl₂, then with HCl in H₂O/dioksan, and finally with dimetilamin in isopropanol afforded the f-acid E-43/HE-50 in 57% yield.^{[61]:24-25,159-172[3]:170-172[5](0:53:17-0:58:30)} Sustained experimentations at Harvard eventually showed the nitrosation method to be successful (N₂O₄, CCl₄, NaOAc ) and to produce the f-carboxyl group even more effectively.^{[3]:172-173[5](0:58:19-0:59:15)}

It was also at Harvard that conditions for the last step were explored, conversion of all remaining ester groups into primary amide groups by ammonolysis. Suyuq ammiak yilda etilen glikol, in the presence of NH₄Cl and the absence of oxygen, converted f-carboxy-hexamethylester E-43/HE-50 into f-carboxy-hexa-amide E-44/HE-51 (= cobyric acid).^{[3]:173-175[53]:24} This was crystallised and shown both as the α-cyano-β-aquo and the α-aquo-β-cyano form to be chromatographically and spectroscopically identical with the corresponding forms of natural cobyric acid.^{[5](0:59:53-1:09:58)[3]:175-176[61]:26-27,196-221} At Harvard, the transformation E-43/HE-50 → E-44/HE-51 was eventually carried out starting with f-amide that had been obtained by umumiy sintez via the A/B approach.^[55]:47-61 The ETH group contented itself with a corresponding f-amide → cobyric acid conversion and subsequent cobyric acid identification where the actual starting material f-amide was derived from vitamin B₁₂.^{[53]:22[61]:15[12]:footnote 45[18]:1570-1571}